ORCID Profile
0000-0003-1380-2347
Current Organisation
Imperial College London
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Publisher: American Society for Clinical Investigation
Date: 03-05-2010
DOI: 10.1172/JCI41560
Publisher: Elsevier BV
Date: 10-2016
Publisher: Oxford University Press (OUP)
Date: 16-05-2019
Abstract: ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of posttranslational decoration by phosphorylcholine (PC). Previously, we showed that ES-62’s PC was attached to N-linked glycans, and using fast atom bombardment mass spectrometry, we characterized the structure of the glycans. However, it was unknown at this time which of ES-62’s four potential N-glycosylation sites carries the PC-modified glycans. In the present study, we now employ more advanced analytical tools—nano-flow liquid chromatography with high-definition electrospray mass spectrometry—to show that PC-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed that up to two PC groups were detected per glycan, and we are now able to characterize N-glycans with up to five PC groups. The number per glycan varies in three of the four glycosylation sites, and in addition, for the first time, we have detected PC on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of PC is detected on terminal GlcNAc, enabling it to interact with the cells and molecules of the immune system. Such expression may explain the potent immunomodulatory effects of a molecule that is considered to have significant therapeutic potential in the treatment of certain human allergic and autoimmune conditions.
Publisher: Public Library of Science (PLoS)
Date: 06-09-2013
Publisher: Elsevier BV
Date: 2010
DOI: 10.1111/J.1538-7836.2009.03665.X
Abstract: von Willebrand factor (VWF) is a key component for maintenance of normal hemostasis. Its glycan moieties, accounting for about 20% of its molecular weight, have been shown to affect many of its properties. Previous studies reported correlations between VWF secretion, half-life and the nature or presence of its N-glycans, and more importantly between VWF plasma level and the type of N-linked ABH antigens. Despite the presence of 10 predicted O-glycosylation sites, the O-glycome remains poorly characterized, impairing the complete elucidation of its influence on VWF functions. So far only a single glycan structure, a disialyl core 1 glycan, has been identified. To define an exhaustive profile of the VWF O-glycan structures to help the understanding of their role in VWF regulation and properties. Plasma-derived VWF O-linked sugars were isolated and analyzed using state-of-the-art mass spectrometry methodologies. We provide here a detailed analysis of the human plasma-derived VWF O-glycome. Eighteen O-glycan structures including both core 1 and core 2 structures are now demonstrated to be present on VWF. Amongst the newly determined structures are unusual tetra-sialylated core 1 O-glycans and ABH antigen-containing core 2 O-glycans. In conjunction with current models explaining VWF activity, knowledge of the complete O-glycome will facilitate research aimed at providing a better understanding of the influence of glycosylation on VWF functions.
Publisher: Springer Science and Business Media LLC
Date: 08-09-2016
DOI: 10.1038/SREP32956
Abstract: The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120 SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens.
Publisher: Portland Press Ltd.
Date: 24-09-2010
DOI: 10.1042/BST0381290
Abstract: Bacteria produce an array of glycan-based structures including capsules, lipo-oligosaccharide and glycosylated proteins, which are invariably cell-surface-located. For pathogenic bacteria, such structures are involved in erse roles in the life cycle of the bacterium, including adhesion, colonization, avoidance of predation and interactions with the immune system. Compared with eukaryotes, bacteria produce huge combinatorial variations of glycan structures, which, coupled to the lack of genetic data, has previously h ered studies on bacterial glycans and their role in survival and pathogenesis. The advent of genomics in tandem with rapid technological improvements in MS analysis has opened a new era in bacterial glycomics. This has resulted in a rich source of novel glycan structures and new possibilities for glycoprospecting and glycoengineering. However, assigning genetic information in predicted glycan biosynthetic pathways to the overall structural information is complex. Bioinformatic analysis is required, linked to systematic mutagenesis and functional analysis of in idual genes, often from erse biosynthetic pathways. This must then be related back to structural analysis from MS or NMR spectroscopy. To aid in this process, systems level analysis of the multiple datasets can be used to make predictions of gene function that can then be confirmed experimentally. The present paper exemplifies these advances with reference to the major gastrointestinal pathogen C ylobacter jejuni.
Publisher: Portland Press Ltd.
Date: 16-03-2023
DOI: 10.1042/BST20221406
Abstract: Protein N-linked glycosylation is a structurally erse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a erse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan–GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan–GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-ecl sia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.
Publisher: Elsevier BV
Date: 10-2015
Publisher: Oxford University Press (OUP)
Date: 25-08-2009
Publisher: Oxford University Press (OUP)
Date: 13-01-2023
Abstract: Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the C ylobacter jejuni N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.
Publisher: Wiley
Date: 07-11-2011
Publisher: Wiley
Date: 27-07-2000
Publisher: Wiley
Date: 15-09-2014
DOI: 10.1111/MMI.12755
Publisher: Elsevier BV
Date: 11-2013
Publisher: American Society for Microbiology
Date: 2011
DOI: 10.1128/JVI.01871-10
Abstract: Although stretches of serine and threonine are sometimes sites for O-linked carbohydrate attachment, specific sequence and structural determinants for O-linked attachment remain ill defined. The gp120 envelope protein of SIVmac239 contains a serine-threonine-rich stretch of amino acids at positions 128 to 139. Here we show that lectin protein from jackfruit seed (jacalin), which binds to non- and monosialylated core 1 O-linked carbohydrate, potently inhibited the replication of SIVmac239. Selection of a jacalin-resistant SIVmac239 variant population resulted in virus with specific substitutions within amino acids 128 to 139. Cloned simian immunodeficiency virus (SIV) variants with substitutions in the 128-to-139 region had infectivities equivalent to, or within 1 log unit of, that of SIVmac239 and were resistant to the inhibitory effects of jacalin. Characterization of the SIVmac239 gp120 O-linked glycome showed the presence of core 1 and core 2 O-linked carbohydrate a 128-to-139-substituted variant gp120 from jacalin-resistant SIV lacked O-linked carbohydrate. Unlike that of SIVmac239, the replication of HIV-1 strain NL4-3 was resistant to inhibition by jacalin. Purified gp120s from four SIVmac and SIVsm strains bound jacalin strongly in an enzyme-linked immunosorbent assay, while nine different HIV-1 gp120s, two SIVcpz gp120s, and 128-to-139-substituted SIVmac239 gp120 did not bind jacalin. The ability or inability to bind jacalin thus correlated with the presence of the serine-threonine-rich stretch in the SIVmac and SIVsm gp120s and the absence of such stretches in the SIVcpz and HIV-1 gp120s. Consistent with sequence predictions, two HIV-2 gp120s bound jacalin, while one did not. These data demonstrate the presence of non- and monosialylated core 1 O-linked carbohydrate on the gp120s of SIVmac and SIVsm and the lack of these modifications on HIV-1 and SIVcpz gp120s.
Publisher: Springer Science and Business Media LLC
Date: 28-06-2008
Publisher: Oxford University Press (OUP)
Date: 20-03-2014
Publisher: American Diabetes Association
Date: 21-02-2011
DOI: 10.2337/DB10-1186
Abstract: Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities. GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured. GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6–sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA. Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.
Publisher: Elsevier BV
Date: 06-2021
Publisher: American Society for Microbiology
Date: 09-2009
DOI: 10.1128/AAC.00389-09
Abstract: DAS181 is a novel candidate therapeutic agent against influenza virus which functions via the mechanism of removing the virus receptor, sialic acid (Sia), from the adjacent glycan structures. DAS181 and its analogues have previously been shown to be potently active against multiple strains of seasonal and avian influenza virus strains in several experimental models, including cell lines, mice, and ferrets. Here we demonstrate that DAS181 treatment leads to desialylation of both α2-6-linked and α2-3-linked Sia in ex vivo human lung tissue culture and primary pneumocytes. DAS181 treatment also effectively protects human lung tissue and pneumocytes against the highly pathogenic avian influenza virus H5N1 (A/Vietnam/3046/2004). Two doses of DAS181 treatment given 12 h apart were sufficient to block H5N1 infection in the ex vivo lung tissue culture. These findings support the potential value of DAS181 as a broad-spectrum therapeutic agent against influenza viruses, especially H5N1.
Publisher: Public Library of Science (PLoS)
Date: 27-06-2017
Publisher: The American Association of Immunologists
Date: 15-08-2006
DOI: 10.4049/JIMMUNOL.177.4.2431
Abstract: Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcα2-6Gal sequence, and a corresponding increase in glycans carrying the Galα1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, α1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.
Publisher: Elsevier BV
Date: 12-2011
Publisher: American Chemical Society (ACS)
Date: 07-08-2018
Publisher: Wiley
Date: 27-08-2013
DOI: 10.1111/IRV.12144
Publisher: Springer Science and Business Media LLC
Date: 15-06-2021
DOI: 10.1038/S41522-021-00220-9
Abstract: Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of ,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.
Publisher: Elsevier BV
Date: 12-2007
DOI: 10.1016/J.JMB.2007.10.042
Abstract: In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.
Publisher: Wiley
Date: 03-2005
Abstract: Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is the pre-eminent technique for mass mapping of glycans. In order to make this technique practical for high-throughput screening, reliable automatic methods of annotating peaks must be devised. We describe an algorithm called Cartoonist that labels peaks in MALDI spectra of permethylated N-glycans with cartoons which represent the most plausible glycans consistent with the peak masses and the types of glycans being analyzed. There are three main parts to Cartoonist. (i) It selects annotations from a library of biosynthetically plausible cartoons. The library we currently use has about 2800 cartoons, but was constructed using only about 300 archetype cartoons entered by hand. (ii) It determines the precision and calibration of the machine used to generate the spectrum. It does this automatically based on the spectrum itself. (iii) It assigns a confidence score to each annotation. In particular, rather than making a binary yes/no decision when annotating a peak, it makes all plausible annotations and associates them with scores indicating the probability that they are correct.
Publisher: Springer Science and Business Media LLC
Date: 04-10-2023
Publisher: Wiley
Date: 05-08-2008
DOI: 10.1111/J.1365-2958.2008.06352.X
Abstract: Proteins in all three domains of life can experience N-glycosylation. The steps involved in the archaeal version of this post-translational modification remain largely unknown. Hence, as the next step in ongoing efforts to identify components of the N-glycosylation pathway of the halophilic archaeon Haloferax volcanii, the involvement of three additional gene products in the biosynthesis of the pentasaccharide decorating the S-layer glycoprotein was demonstrated. The genes encoding AglF, AglI and AglG are found immediately upstream of the gene encoding the archaeal oligosaccharide transferase, AglB. Evidence showing that AglF and AglI are involved in the addition of the hexuronic acid found at position three of the pentasaccharide is provided, while AglG is shown to contribute to the addition of the hexuronic acid found at position two. Given their proximities in the H. volcanii genome, the transcription profiles of aglF, aglI, aglG and aglB were considered. While only aglF and aglI share a common promoter, transcription of the four genes is co-ordinated, as revealed by determining transcript levels in H. volcanii cells raised in different growth conditions. Such changes in N-glycosylation gene transcription levels offer additional support for the adaptive role of this post-translational modification in H. volcanii.
Publisher: Elsevier BV
Date: 2022
Publisher: Elsevier BV
Date: 2023
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.EXPPARA.2016.04.012
Abstract: Glycoconjugates play a crucial role in the host-parasite relationships of helminthic infections, including angiostrongyliasis. It has previously been shown that the antigenicity of proteins from female Angiostrongylus cantonensis worms may depend on their associated glycan moieties. Here, an N-glycan profile of A. cantonensis is reported. A total soluble extract (TE) was prepared from female A. cantonensis worms and was tested by western blot before and after glycan oxidation or N- and O-glycosidase treatment. The importance of N-glycans for the immunogenicity of A. cantonensis was demonstrated when deglycosylation of the TE with PNGase F completely abrogated IgG recognition. The TE was also fractionated using various lectin columns [Ulex europaeus (UEA), concanavalin A (Con A), Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Lycopersicon esculentum (LEA)], and then each fraction was digested with PNGase F. Released N-glycans were analyzed with matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) and MALDI-TOF/TOF-MS/MS. Complex-type, high mannose, and truncated glycan structures were identified in all five fractions. Sequential MALDI-TOF-TOF analysis of the major MS peaks identified complex-type structures, with a α1-6 fucosylated core and truncated antennas. Glycoproteins in the TE were labeled with BodipyAF558-SE dye for a lectin microarray analysis. Fluorescent images were analyzed with ProScanArray imaging software followed by statistical analysis. A total of 29 lectins showed positive binding to the TE. Of these, Bandeiraea simplicifolia (BS-I), PNA, and Wisteria floribunda (WFA), which recognize galactose (Gal) and N-acetylgalactosamine (GalNAc), exhibited high affinity binding. Taken together, our findings demonstrate that female A. cantonensis worms have characteristic helminth N-glycans.
Publisher: Cold Spring Harbor Laboratory
Date: 24-11-2021
DOI: 10.1101/2021.11.24.21266804
Abstract: Mutations in the FUT2 gene that result in a lack of expression of histo-blood group antigens on secreted glycoproteins may shape the vaginal microbiota with consequences for birth outcome. To test this, we analysed the relationship between secretor status, vaginal microbiota and gestational length in an ethnically erse cohort of 313 pregnant women, including 91 who delivered prematurely. Lactobacillus species were found to co-occur less often with other microbial taxa in non-secretors. Moreover, non-secretors with Lactobacillus spp. depleted vaginal microbiota in early pregnancy had significantly shorter gestational length than Lactobacillus spp. dominated non-secretors (mean of 245.5 (SD=44.5) versus 265.9 (23.6)) p=0.045), but not compared to Lactobacillus spp. dominated (261.8 (27.5)) and depleted (264.3 days (21.2)) secretors. In identifying a relationship between blood-group antigen expression and vaginal microbiota-host interactions, our results point towards stratification by secretor status as an important factor for considering preterm birth risk and prevention.
Publisher: American Society for Microbiology
Date: 10-2017
DOI: 10.1128/JVI.01228-17
Abstract: A highly conserved threonine near the C terminus of gp120 of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) was investigated for its contributions to envelope protein function and virion infectivity. When this highly conserved Thr residue was substituted with anything other than serine (the other amino acid that can accept O-glycosylation), the resulting virus was noninfectious. We found that this Thr was critical for the association of gp120 with the virion and that amino acid substitution increased the amount of dissociated gp120 in the cell culture supernatant. When HIV virions were generated in cells overexpressing polypeptide N -acetylgalactosaminyltransferase 1 (GalNAcT1), viral infectivity was increased 2.5-fold compared to that of virus produced in wild-type HEK293T cells infectivity was increased 8-fold when the Thr499Ser mutant was used. These infectivity enhancements were not observed when GalNAcT3 was used. Using HEK293T knockout cell lines totally devoid of the ability to perform O-linked glycosylation, we demonstrated production of normal levels of virions and normal levels of infectivity in the complete absence of O-linked carbohydrate. Our data indicate that O-glycosylation is not necessary for the natural replication cycle of HIV and SIV. Nonetheless, it remains theoretically possible that the repertoire of GalNAc transferase isoforms in natural target cells for HIV and SIV in vivo could result in O-glycosylation of the threonine residue in question and that this could boost the infectivity of virions beyond the levels seen in the absence of such O-glycosylation. IMPORTANCE Approximately 50% of the mass of the gp120 envelope glycoprotein of both HIV and SIV is N-linked carbohydrate. One of the contributions of this N-linked carbohydrate is to shield conserved peptide sequences from recognition by humoral immunity. This N-linked glycosylation is one of the reasons that primary isolates of HIV and SIV are so heavily resistant to antibody-mediated neutralization. Much less studied is any potential contribution from O-linked glycosylation. The literature on this topic to date is somewhat confusing and ambiguous. Our studies described in this report demonstrate unambiguously that O-linked glycosylation is not necessary for the natural replication cycle of HIV and SIV. However, the door is not totally closed because of the ersity of numerous GalNAc transferase enzymes that initiate O-linked carbohydrate attachment and the theoretical possibility that natural target cells for HIV and SIV in vivo could potentially complete such O-linked carbohydrate attachment to further increase infectivity.
Publisher: Wiley
Date: 02-2010
DOI: 10.1111/J.1365-2958.2009.07045.X
Abstract: Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.
Publisher: American Society for Clinical Investigation
Date: 27-04-2015
DOI: 10.1172/JCI59987
Publisher: Proceedings of the National Academy of Sciences
Date: 22-09-2009
Abstract: Millions afflicted with Chagas disease and other disorders of aberrant glycosylation suffer symptoms consistent with altered electrical signaling such as arrhythmias, decreased neuronal conduction velocity, and hyporeflexia. Cardiac, neuronal, and muscle electrical signaling is controlled and modulated by changes in voltage-gated ion channel activity that occur through physiological and pathological processes such as development, epilepsy, and cardiomyopathy. Glycans attached to ion channels alter channel activity through isoform-specific mechanisms. Here we show that regulated and aberrant glycosylation modulate cardiac ion channel activity and electrical signaling through a cell-specific mechanism. Data show that nearly half of 239 glycosylation-associated genes (glycogenes) were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. The N-glycan structures produced among cardiomyocyte types were markedly variable. Thus, the cardiac glycome, defined as the complete set of glycan structures produced in the heart, is remodeled. One glycogene, ST8sia2, a polysialyltransferase, is expressed only in the neonatal atrium. Cardiomyocyte electrical signaling was compared in control and ST8sia2 (−/−) neonatal atrial and ventricular myocytes. Action potential waveforms and gating of less sialylated voltage-gated Na + channels were altered consistently in ST8sia2 (−/−) atrial myocytes. ST8sia2 expression had no effect on ventricular myocyte excitability. Thus, the regulated (between atrium and ventricle) and aberrant (knockout in the neonatal atrium) expression of a single glycogene was sufficient to modulate cardiomyocyte excitability. A mechanism is described by which cardiac function is controlled and modulated through physiological and pathological processes that involve regulated and aberrant glycosylation.
Publisher: Public Library of Science (PLoS)
Date: 27-04-2015
Publisher: American Physiological Society
Date: 05-2011
Abstract: N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl 4 were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl 4 models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl 4 model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.
Publisher: Public Library of Science (PLoS)
Date: 20-04-2015
Publisher: Elsevier BV
Date: 2007
Publisher: Elsevier BV
Date: 10-2011
Publisher: Hindawi Limited
Date: 2010
DOI: 10.1155/2010/754101
Abstract: Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L 1004 - Q 1395 . Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Gl c 1 Ma n 2 GlcNA c 2 plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b 558 / 566 of S. acidocaldarius . S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya .
Publisher: Elsevier BV
Date: 02-2016
Publisher: Springer Science and Business Media LLC
Date: 19-03-2021
DOI: 10.1038/S41467-021-21814-Z
Abstract: In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man 5-9 GlcNAc 2 ), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum -infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.
Publisher: Elsevier BV
Date: 03-2012
Publisher: Portland Press Ltd.
Date: 26-09-2012
DOI: 10.1042/BJ20120810
Abstract: vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is le evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of in iduals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn2635 site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.
Publisher: Portland Press Ltd.
Date: 09-1975
DOI: 10.1042/BJ1490754
Publisher: American Society for Microbiology
Date: 10-2010
DOI: 10.1128/JB.00211-10
Abstract: The first bacterial N-linked glycosylation system was discovered in C ylobacter jejuni , and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria , including three Helicobacter species: H. pullorum , H. canadensis , and H. winghamensis . In contrast to C. jejuni , in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes ( pglB1 and pglB2 ), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli . Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum , PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the −2 position of the N-glycosylation sequon, as for C. jejuni . Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.
Publisher: American Society for Microbiology
Date: 15-04-2009
DOI: 10.1128/JB.01406-08
Abstract: Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycosylation and lipopolysaccharide (LPS) O-antigen biosynthesis. This island appears to have been laterally acquired as it is flanked by insertion element-like sequences and has a much lower G+C content than the average aeromonad G+C content. Most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation in both C ylobacter jejuni and Helicobacter pylori . Two of the genes, lst and lsg , are LPS specific as mutation of them results in the loss of only a band for the LPS O-antigen. Lsg encodes a putative Wzx flippase, and mutation of Lsg affects only LPS this finding supports the notion that flagellin glycosylation occurs within the cell before the flagellins are exported and assembled and not at the surface once the sugar has been exported. The proteins encoded by flmA , flmB , neuA , flmD , and neuB are thought to make up a pseudaminic acid biosynthetic pathway, and mutation of any of these genes resulted in the loss of motility, flagellar expression, and a band for the LPS O-antigen. Furthermore, pseudaminic acid was shown to be present on both flagellin subunits that make up the polar flagellum filament, to be present in the LPS O-antigen of the A. caviae wild-type strain, and to be absent from the A. caviae flmD mutant strain.
Publisher: Elsevier BV
Date: 12-2014
Publisher: MDPI AG
Date: 16-10-2015
DOI: 10.3390/BIOM5042758
Publisher: Oxford University Press (OUP)
Date: 03-04-2006
Abstract: The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1 however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.
Publisher: Elsevier BV
Date: 08-2020
Publisher: American Chemical Society (ACS)
Date: 17-12-2005
DOI: 10.1021/BI0512804
Abstract: Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.
Publisher: Oxford University Press (OUP)
Date: 19-07-2007
Abstract: CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.
Publisher: American Association for Cancer Research (AACR)
Date: 04-2012
DOI: 10.1158/1538-7445.AM2012-3417
Abstract: Background When breast carcinomas remain confined to breast tissue, cure rates exceed 90% (sr/1975_2006/). However, if the cancer disseminates through the body, long-term survival decreases depending upon the extent of, and the sites of, colonisation Genes that control the different stages of the metastatic process need to be identified to better delineate the process, and to aid in the development of metastatic biomarkers and provide potential targets for the treatment of metastatic disease. Genetic screens, such as those that exploit RNA interference (RNAi), provide an unbiased approach to the identification of genes associated with a phenotype of interest. Although cell-based screens have been highly informative in identifying genes involved in tumour cell survival, migration and invasion, these in vitro approaches are largely unsuitable for interrogating the later stages of the metastatic process, in particular the processes of cell dissemination, tumour cell extravasation from the circulation and colonisation of secondary sites. Method Using the 4T1 mouse model an in vivo functional metastasis screen that integrates RNAi technology and massively parallel sequencing was implemented to rapidly discover and validate metastasis genes. Results Using this approach, 12 ‘hits’ that suppress tumour cell colonisation of the lungs were identified. 3 of the top 5 hits have been validated as novel metastasis suppressor genes in both mouse and human cell lines and further studies have been undertaken to elucidate the mechanism of action of the top hit. Critically, these findings are clinically relevant in primary breast cancers where there is a significant correlation between elevated expression levels of these suppressor genes and reduced frequency of metastatic events. Conclusion This study demonstrates the value of adopting an unbiased methodology to discover novel metastatic genes and establishes, for the first time, that an in vivo metastasis screen can be combined with next generation sequencing to identify novel components of the metastatic process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research 2012 Mar 31-Apr 4 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2012 (8 Suppl):Abstract nr 3417. doi:1538-7445.AM2012-3417
Publisher: MDPI AG
Date: 25-03-2022
DOI: 10.3390/MICROORGANISMS10040708
Abstract: Brucellosis is a global disease and the world’s most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.
Publisher: American Society for Microbiology
Date: 15-05-2013
DOI: 10.1128/JB.00035-13
Abstract: Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man 2 Glc 1 GlcNAc 2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16 , coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N -glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N -glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δ agl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δ agl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N -glycan size.
Publisher: Springer Science and Business Media LLC
Date: 06-2018
DOI: 10.1007/S10719-018-9825-8
Abstract: Glycosylation is considered one of the most complex and structurally erse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious s le preparation steps. Therefore, in this study, we aim to improve s le preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein rotein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of s les proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.
Publisher: American Chemical Society (ACS)
Date: 30-08-2007
DOI: 10.1021/PR070239F
Abstract: We describe Peptoonist, a program that can automatically identify the glycans (sugars) present at each N-glycosylation site of a protein. The input to Peptoonist is a series of mass spectra, both MS and MS/MS, obtained from a liquid chromatography (LC) run of proteolytically digested purified glycoproteins. The program uses MS/MS to identify glycosylated peptides and single-MS to identify the N-glycans present on each of these peptides, at least to the level of monosaccharide composition. We validate the program on an LC run of mouse zona pellucida proteins that had been intensively hand annotated by a human expert. Our program doubled the number of glycopeptide identifications, and also found several possible errors in the hand annotation. In addition, it automatically made most of the same glycan isomer identifications as the expert annotator.
Publisher: Wiley
Date: 09-05-2021
DOI: 10.1111/IMM.13341
Abstract: Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system–immune interface. Using organotypic cerebellar slice cultures (OSCs) from transgenic mice, we induced extensive immune‐mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of green fluorescent protein expression, immunohistochemistry and confocal microscopy. Cysteine‐ and glycan‐adapted Fc fragments protected OSC from demyelination in a dose‐dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement‐mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together, our findings show that recombinant biomimetics can be made that are at least two hundred‐fold more effective than IVIG in controlling demyelination by anti‐MOG antibodies.
Publisher: Oxford University Press (OUP)
Date: 10-07-2007
Abstract: Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size. In this paper, we describe MALDI strategies that have been optimized for the analysis of highly sulfated GAG-derived oligosaccharides. A crystalline matrix norharmane, as well as an ionic liquid 1-methylimidazolium alpha-cyano-4-hydroxycinnamate (ImCHCA), have been used for the analysis of heparin di-, tetra-, hexa- and decasaccharides carrying from 2 to 13 sulfate groups. Information about the maximum number of sulfate groups is obtained using the ionic liquid whereas MALDI-TOF/TOF MS/MS experiments using norharmane allowed the determination of the nature of the glycosidic backbone, and more precise information about the presence and the position in the sequence of N-acetylated residues.
Publisher: MDPI AG
Date: 14-03-2014
DOI: 10.3390/IJMS15034492
Publisher: Oxford University Press (OUP)
Date: 07-2011
Publisher: Public Library of Science (PLoS)
Date: 06-07-2015
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.BBRC.2010.11.005
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 amino-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single in iduals, and that in iduals of two different ethnicities possess a similar PCI pattern, verifying that the micro-heterogeneity is conserved among humans. Furthermore, we have provided experimental evidence that PCI in both in iduals is O-glycosylated on Thr20 with a core type 1 O-glycan, which is mostly NeuAcGalGalNAc. Modeling suggested that the O-glycan attachment site is located in proximity to several ligand-binding sites of the inhibitor.
Publisher: Public Library of Science (PLoS)
Date: 15-07-2010
Publisher: Wiley
Date: 29-03-2010
DOI: 10.1111/J.1365-2958.2010.07090.X
Abstract: While pathways for N-glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post-translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S-layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N-glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S-adenosyl-L-methionine-dependent methyltransferase.
Publisher: Public Library of Science (PLoS)
Date: 14-03-2013
Publisher: Elsevier
Date: 2010
Publisher: Elsevier BV
Date: 12-1997
DOI: 10.1016/S1367-5931(97)80047-6
Abstract: Early magnetic resonance imaging (MRI) for acute low back pain (LBP) has been associated with increased costs, greater health care utilization, and longer disability duration in workers' compensation claimants. To assess the impact of a state policy implemented in June 2010 that required prospective utilization review (UR) for early MRI among workers' compensation claimants with LBP. Interrupted time series. In total, 76,119 Washington State workers' compensation claimants with LBP between 2006 and 2014. Proportion of workers receiving imaging per month (MRI, computed tomography, radiographs) and lumbosacral injections and surgery mean total health care costs per worker mean duration of disability per worker. Measures were aggregated monthly and attributed to injury month. After accounting for secular trends, decreases in early MRI [level change: -5.27 (95% confidence interval, -4.22 to -6.31) trend change: -0.06 (-0.01 to -0.12)], any MRI [-4.34 (-3.01 to -5.67) -0.10 (-0.04 to -0.17)], and injection [trend change: -0.12 (-0.06 to -0.18)] utilization were associated with the policy. Radiograph utilization increased in parallel [level change: 2.46 (1.24-3.67)]. In addition, the policy resulted in significant decreasing changes in mean costs per claim, mean disability duration, and proportion of workers who received disability benefits. The policy had no effect on computed tomography or surgery utilization. The UR policy had discernable effects on health care utilization, costs, and disability. Integrating evidence-based guidelines with UR can improve quality of care and patient outcomes, while reducing use of low-value health services.
Publisher: Elsevier BV
Date: 10-2010
Publisher: American Society for Microbiology
Date: 11-2010
DOI: 10.1128/JB.00705-10
Abstract: Like the Eukarya and Bacteria , the Archaea also perform N glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO _ 1517 to agl genes encoding known elements of the H. volcanii N-glycosylation process. By combining the deletion of HVO _ 1517 with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of aglJ , however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while aglJ can be deleted, H. volcanii surface layer integrity is compromised in the absence of the encoded protein.
Publisher: Elsevier BV
Date: 05-2006
Publisher: Elsevier BV
Date: 03-2023
DOI: 10.1016/J.JID.2022.07.033
Abstract: The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TIC) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines (poly-LacNAc) via loss of I-branching enzyme, β1,6 N-acetylglucosaminyltransferase 2 (GCNT2). Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. Here, we explored hypoxia-dependent alterations in MM glycome-associated genes and found that GCNT2 was downregulated and a galectin (Gal)-8 - ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low GCNT2 levels correlated with poor patient outcome and patient serum s les were elevated for Gal-8. Depressed GCNT2 in MM cells upregulated TIC marker, nerve growth factor receptor (NGFR)/CD271, whereas loss of MM cell-intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-LacNAcs on N-glycans of the TIC marker and pro-metastatic molecule CD44, among other receptors and activated pro-survival factor AKT. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-LacNAc and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-LacNAcs and Gal-8 as biomarkers and therapeutic targets of MM.
Publisher: Oxford University Press (OUP)
Date: 31-10-2016
Publisher: The American Association of Immunologists
Date: 15-12-2007
DOI: 10.4049/JIMMUNOL.179.12.8216
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.
Publisher: Springer Science and Business Media LLC
Date: 17-12-2008
Publisher: Oxford University Press (OUP)
Date: 10-12-2008
DOI: 10.1093/BIOINFORMATICS/BTN636
Abstract: Motivation: In the past few years, mass spectrometry (MS) has emerged as the premier tool for identification and quantification of biological molecules such as peptides and glycans. There are two basic strategies: single-MS, which uses a single round of mass analysis, and MS/MS (or higher order MSn), which adds one or more additional rounds of mass analysis, interspersed with fragmentation steps. Single-MS offers higher throughput, broader mass coverage and more direct quantitation, but generally much weaker identification. Single-MS, however, does work fairly well for the case of N-glycan identification, which are more constrained than other biological polymers. We previously demonstrated single-MS identification of N-glycans to the level of ‘cartoons’ (monosaccharide composition and topology) by a system that incorporates an expert's detailed knowledge of the biological s le. In this article, we explore the possibility of ab initio single-MS N-glycan identification, with the goal of extending single-MS, or primarily-single-MS, identification to non-expert users, novel conditions and unstudied tissues. Results: We propose and test three cartoon-assignment algorithms that make inferences informed by biological knowledge about glycan synthesis. To test the algorithms, we used 71 single-MS spectra from a variety of tissues and organisms, containing more than 2800 manually annotated peaks. The most successful of the algorithms computes the most richly connected subgraph within a ‘cartoon graph’. This algorithm uniquely assigns the correct cartoon to more than half of the peaks in 41 out of the 71 spectra. Contact: goldberg@parc.com Supplementary information: Supplementary data are available at Bioinformatics online.
Publisher: Wiley
Date: 14-01-2020
DOI: 10.1111/FEBS.15197
Publisher: Oxford University Press (OUP)
Date: 22-11-2016
Publisher: Public Library of Science (PLoS)
Date: 17-03-2017
Publisher: American Association for Cancer Research (AACR)
Date: 04-2012
DOI: 10.1158/1538-7445.AM2012-2316
Abstract: Itraconazole is a widely used antifungal drug that has been recently shown to inhibit angiogenesis in vivo and to block non-small cell lung cancer growth in animal models. Based on these and other results, itraconazole is currently being evaluated in four clinical trials as a cancer therapeutic. Angiogenesis, one of the hallmarks of cancer, is required for tumor growth and is driven by tumor-secreted growth factors including vascular endothelial growth factor (VEGF). We now report that in endothelial cells, itraconazole inhibits signaling of VEGF through the VEGF receptor 2 (VEGFR2) (80% inhibition p & 0.05) and subsequently through a downstream substrate, PLCϒ1 (60% inhibition p & 0.05). Mechanistically, we show that this was due to a nearly complete loss of VEGF binding to VEGR2 that was secondary to a loss of cell surface expression of the receptor. After itraconazole treatment, VEGFR2 was retained in a perinuclear aggregate which partially overlapped with the cis-Golgi marker GM130. Correlated with this loss of VEGFR2 signaling and trafficking, was a dramatic change in VEGFR2 mobility on SDS-PAGE which resulted from incomplete N-glycosylation. Itraconazole globally reduced poly-LacNAc and tetra-antennary glycans in endothelial cells and induced hypoglycosylation of EGFR in a renal cell carcinoma line, suggesting that the effects of itraconazole apply to multiple cell types and receptors. Importantly, small molecule inducers of lysosomal accumulation and mTOR inhibition, two previously known itraconazole-induced effects, did not interfere with VEGFR2 glycosylation. Similarly, glycosylation inhibitors did not affect cholesterol trafficking or inhibit mTOR. However, increasing cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, restored VEGFR2 glycosylation and signaling. Thus, the newly described properties of itraconazole occur in parallel to inhibition of cholesterol trafficking and mTOR but appear downstream of a single target. Taking these findings together, we propose a model in which itraconazole inhibits intracellular trafficking. This model could explain the loss of VEGFR2 signaling, incomplete VEGFR2 glycosylation, as well as disruption of cholesterol trafficking and mTOR signaling. Importantly, the effects of itraconazole we report here may significantly contribute to the in vivo efficacy of itraconazole and are relevant to ongoing clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research 2012 Mar 31-Apr 4 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2012 (8 Suppl):Abstract nr 2316. doi:1538-7445.AM2012-2316
Publisher: Wiley
Date: 28-02-2022
DOI: 10.1002/BIT.28066
Abstract: Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half‐life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream–downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc γ‐based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space to establish holistic and consistent manufacturing processes for the production of antibody therapies.
Publisher: Springer Science and Business Media LLC
Date: 18-08-2023
DOI: 10.1186/S12934-023-02125-Y
Abstract: Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N- oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N- OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli . The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.
Publisher: Microbiology Society
Date: 06-2005
Abstract: The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium – intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis , produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis . In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis , suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N -acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum , M. peregrinum , M. chelonae and M. abscessus .
Publisher: Oxford University Press (OUP)
Date: 15-05-2006
Abstract: The white-tailed deer is the definitive host of the parasitic nematode Parelaphostrongylus tenuis. This parasite also infects a wide variety of domesticated livestock, causing a debilitating neurologic disease. Glycoconjugates are becoming increasingly implicated in nematode strategies to maintain persistent infections in immunologically competent hosts. In this study, we have carried out detailed mass spectrometric analysis together with classical biochemical techniques, including western blotting and immunohistochemical staining with anticarbohydrate monoclonal antibodies and have shown that P. tenuis contains complex-type N-glycans with the antennae capped with Galalpha1-3Galbeta1-4GlcNAc sequence. By mimicking a vertebrate glycan, Galalpha1-3Gal may aid the parasite in evading immunological detection by the host. This is the first report of the Galalpha1-3Gal sequence in a nematode.
Publisher: Oxford University Press (OUP)
Date: 20-09-2010
Publisher: Elsevier BV
Date: 02-2020
Publisher: Elsevier BV
Date: 12-2016
Publisher: Elsevier BV
Date: 07-2011
Publisher: Wiley
Date: 04-02-2005
DOI: 10.1111/J.1365-2958.2005.04519.X
Abstract: We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen C ylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five in idual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.
Publisher: Elsevier BV
Date: 12-2016
Publisher: Mary Ann Liebert Inc
Date: 15-09-2020
Publisher: Elsevier BV
Date: 12-2005
Publisher: The Royal Society
Date: 10-2020
DOI: 10.1098/RSOB.200218
Abstract: Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum ( Trichosurus vulpecula ) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.
Publisher: Frontiers Media SA
Date: 14-12-2018
Publisher: Wiley
Date: 26-10-2007
DOI: 10.1002/IJC.22958
Abstract: We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis(x/a) antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the beta-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the beta-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcbeta4(NeuAcalpha3)Galbeta3(NeuAcalpha6)GlcNAcbetaGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Mono- and disialyl core Type 1 O-linked structures were identified after reductive beta-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O-glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its beta-chain are characterized by enhanced branching as well as antenna fucosylation.
Publisher: Elsevier BV
Date: 02-2010
Publisher: Proceedings of the National Academy of Sciences
Date: 07-2011
Abstract: Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca 2+ . As an “adhesion G-protein-coupled receptor,” LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, we used LPH1’s extracellular domain to purify its endogenous ligand. A single protein of ∼275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. We show that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippoc al neurons in cocultures LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippoc al neurons show that LPH1 and Lasso are enriched in synapses in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca 2+ signals in presynaptic boutons of hippoc al neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca 2+ signals, which might affect synaptic functions.
Publisher: Public Library of Science (PLoS)
Date: 11-02-2020
Publisher: Proceedings of the National Academy of Sciences
Date: 10-10-2018
Abstract: Diverse polysaccharides are installed on specific asparagine residues as glycoproteins traverse the endoplasmic reticulum and Golgi. These N-glycan structures comprise the N-glycome, which coats cell surfaces, regulates cell–cell and cell–matrix interactions, and has functional consequences for immune system function and beyond. Our understanding of how intracellular signaling regulates the molecular architecture of the N-glycome remains immature. We show that the transcription factor XBP1s alters N-glycan structures displayed on endogenous membrane-associated and secreted glycoproteins, coincident with XBP1s-induced changes in N-glycosylation–related transcripts. These results establish a role for the unfolded protein response in defining the global composition of the N-glycome–providing a mechanism for transducing internal stress to an external signal, a phenomenon with implications for both normal biology and pathology.
Publisher: Oxford University Press (OUP)
Date: 19-10-2005
Abstract: Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes, we employed the Affymetrix technology to develop a focused and highly annotated glycogene-chip representing human and murine glycogenes, including glycosyltransferases, nucleotide sugar transporters, glycosidases, proteoglycans, and glycan-binding proteins. In this report, the array has been used to generate glycogene-expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm reveals that expression profiles in immune tissues (thymus [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell-type-specific glycosylation. Comparison of gene expression profiles with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and Siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (CFG) (www.functionalglycomics.org).
Publisher: Springer Science and Business Media LLC
Date: 19-12-2015
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-09-2011
Abstract: Fertilization in humans is initiated by binding of spermatozoa to a selectin ligand on the egg’s extracellular matrix.
Publisher: American Chemical Society (ACS)
Date: 26-11-2013
DOI: 10.1021/JA409781C
Publisher: Mary Ann Liebert Inc
Date: 11-2013
Publisher: Springer Science and Business Media LLC
Date: 22-08-2018
DOI: 10.1038/S41467-018-05795-0
Abstract: Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.
Publisher: Microbiology Society
Date: 07-2010
Abstract: The C ylobacter jejuni flagellin protein is O- glycosylated with structural analogues of the nine-carbon sugar pseudaminic acid. The most common modifications in the C. jejuni 81-176 strain are the 5,7-di- N- acetylated derivative (Pse5Ac7Ac) and an acetamidino-substituted version (Pse5Am7Ac). Other structures detected include O- acetylated and N- acetylglutamine-substituted derivatives (Pse5Am7Ac8OAc and Pse5Am7Ac8GlnNAc, respectively). Recently, a derivative of pseudaminic acid modified with a di- O- methylglyceroyl group was detected in C. jejuni NCTC 11168 strain. The gene products required for Pse5Ac7Ac biosynthesis have been characterized, but those genes involved in generating other structures have not. We have demonstrated that the mobility of the NCTC 11168 flagellin protein in SDS-PAGE gels can vary spontaneously and we investigated the role of single nucleotide repeats or homopolymeric-tract-containing genes from the flagellin glycosylation locus in this process. One such gene, Cj1295, was shown to be responsible for structural changes in the flagellin glycoprotein. Mass spectrometry demonstrated that the Cj1295 gene is required for glycosylation with the di- O- methylglyceroyl-modified version of pseudaminic acid.
Publisher: American Chemical Society (ACS)
Date: 08-2004
DOI: 10.1021/BI049622D
Abstract: Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.
Publisher: Wiley
Date: 2009
DOI: 10.1111/J.1365-2958.2008.06536.X
Abstract: Previously mutations in a putative protein O-mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, phiC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt(-) derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor. A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C(45)-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo.
Publisher: Springer Science and Business Media LLC
Date: 12-08-2010
Abstract: There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model. Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory. In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. Conclusions : We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.
Publisher: American Society for Microbiology
Date: 15-02-2016
DOI: 10.1128/JVI.01722-15
Abstract: Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein β-1,6- N -acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O -glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus. IMPORTANCE Viruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme which is a key enzyme for the synthesis of complex O -glycans. In this study, we show that, in contrast to cellular homologues, this virus has evolved to alternatively express two proteins from this gene. While the first one is enzymatically active, the second results from the alternative splicing of the region encoding the catalytic site of the enzyme. We postulate that this regulatory mechanism could allow the virus to modulate the synthesis of some particular glycans for function at the location and/or the moment of infection.
Publisher: Shared Science Publishers OG
Date: 05-2017
Publisher: Portland Press Ltd.
Date: 24-09-2010
DOI: 10.1042/BST0381307
Abstract: With glycosylation now firmly established across both Archaeal and bacterial proteins, a wide array of glycan ersity has become evident from structural analysis and genomic data. These discoveries have been built in part on the development and application of mass spectrometric technologies to the bacterial glycoproteome. This review highlights recent findings using high sensitivity MS of the large variation of glycans that have been reported on flagellin and pilin proteins of bacteria, using both ‘top down’ and ‘bottom up’ approaches to the characterization of these glycoproteins. We summarize current knowledge of the sugar modifications that have been observed on flagellins and pilins, in terms of both the erse repertoire of monosaccharides observed, and the assemblage of moieties that decorate many of these sugars.
Publisher: Oxford University Press (OUP)
Date: 11-06-2019
Abstract: The Symbol Nomenclature for Glycans (SNFG) is a community-curated standard for the depiction of monosaccharides and complex glycans using various colored-coded, geometric shapes, along with defined text additions. It is hosted by the National Center for Biotechnology Information (NCBI) at the NCBI-Glycans Page (lycans/snfg.html). Several changes have been made to the SNFG page in the past year to update the rules for depicting glycans using the SNFG, to include more ex les of use, particularly for non-mammalian organisms, and to provide guidelines for the depiction of ambiguous glycan structures. This Glycoforum article summarizes these recent changes.
Publisher: The American Association of Immunologists
Date: 11-2007
DOI: 10.4049/JIMMUNOL.179.9.5701
Abstract: To fulfil their function as APCs, dendritic cells (DC) and their precursors need to travel from blood to the peripheral tissues and, upon activation, migrate from tissues to draining lymph nodes. Because O-glycans play a role in T cell trafficking, we investigated the O-glycosylation profile of human monocyte-derived DC. Sialyl-Lewisx (sLex), a glycan involved in extravasation via selectin binding, was found to be expressed exclusively on P-selectin glycoprotein ligand-1 in monocytes and immature DC. However, sLex was lost from mature DC even though these cells retained expression of P-selectin glycoprotein ligand-1. Maturation of DC led to a rapid change in the expression of glycosyltransferases involved in O-linked glycosylation. A down-regulation of C2GnT1 mRNA and enzymatic activity was observed with a concurrent up-regulation of ST3Gal I and ST6GalNAc II mRNA resulting in a loss of the core 2 structures required for sLex expression as a P-selectin ligand. Interestingly, the early regulation of these glycosyltransferases was mediated by PGE2, which is known to be required for human DC migration. The pattern of O-glycosylation seen in mature cells was very similar to that expressed by naive T cells, which home to lymph nodes. Our data show that the regulation of O-glycosylation controls sLex expression, and also suggest that O-glycans may have a function in DC migration.
Publisher: American Society for Microbiology
Date: 05-2008
DOI: 10.1128/JB.00056-08
Abstract: Archaea , like Eukarya and Bacteria , are able to N glycosylate select protein targets. However, in contrast to relatively advanced understanding of the eukaryal N glycosylation process and the information being amassed on the bacterial process, little is known of this posttranslational modification in Archaea . Toward remedying this situation, the present report continues ongoing efforts to identify components involved in the N glycosylation of the Haloferax volcanii S-layer glycoprotein. By combining gene deletion together with mass spectrometry, AglE, originally identified as a homologue of murine Dpm1, was shown to play a role in the addition of the 190-Da sugar subunit of the novel pentasaccharide decorating the S-layer glycoprotein. Topological analysis of an AglE-based chimeric reporter assigns AglE as an integral membrane protein, with its N terminus and putative active site facing the cytoplasm. These finding, therefore, contribute to the developing picture of the N glycosylation pathway in Archaea .
Publisher: Elsevier BV
Date: 2013
Publisher: Walter de Gruyter GmbH
Date: 11-10-2012
Abstract: During the EUROCarbDB project our group developed the GlycanBuilder and GlycoWorkbench glycoinformatics tools. This short communication summarizes the capabilities of these two tools and updates which have been made since the original publications in 2007 and 2008. GlycanBuilder is a tool that allows for the fast and intuitive drawing of glycan structures this tool can be used standalone, embedded in web pages and can also be integrated into other programs. GlycoWorkbench has been designed to semi-automatically annotate glycomics data. This tool can be used to annotate mass spectrometry (MS) and MS/MS spectra of free oligosaccharides, N and O-linked glycans, GAGs ( g lycos a mino g lycans) and glycolipids, as well as MS spectra of glycoproteins.
Publisher: Springer Science and Business Media LLC
Date: 07-08-2007
Publisher: Elsevier BV
Date: 07-2008
Publisher: Elsevier BV
Date: 04-2010
Publisher: Elsevier BV
Date: 2008
Publisher: Portland Press Ltd.
Date: 21-09-2011
DOI: 10.1042/BST0391334
Abstract: N-glycans are key players mediating cell–cell communication in the immune system, interacting with glycan-binding proteins. In the present article, we discuss key themes that are emerging from the structural analysis of complex-type N-linked glycans from human and murine immune cell lines, employing high-sensitivity MALDI (matrix-assisted laser desorption ionization)–TOF (time-of-flight) MS technology. Particular focus is given to terminal epitopes, the abundance of multiply branched N-glycans and how glycosylation can affect human health in diseases such as congenital neutropenia and glycogen storage disease.
Publisher: Elsevier BV
Date: 04-2005
Publisher: Elsevier BV
Date: 02-2006
DOI: 10.1016/J.VACCINE.2005.08.075
Abstract: Francisella tularensis live vaccine strain (LVS) produces two colony types when grown on solid media, often referred to as blue variants (BV) and grey variants (GV). Whereas blue variant bacteria possessed a lipopolysaccharide O-side chain, grey variant bacteria lacked O-side chains. Grey variant bacteria appeared in stationary phase bacterial cultures and could be identified using a novel FACS-based assay. Compared to blue variant bacteria, grey variants showed a reduced ability to infect and survive in macrophages. The immunisation of mice with blue variant bacteria, but not grey variant bacteria, induced protective immunity towards fully virulent F. tularensis.
Publisher: Spandidos Publications
Date: 19-11-2009
DOI: 10.3892/IJO_00000490
Publisher: Springer Science and Business Media LLC
Date: 07-2006
DOI: 10.1007/S10719-006-6693-4
Abstract: With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galbeta1-4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galbeta1-3GalNAc sequence.
Publisher: Springer Science and Business Media LLC
Date: 15-07-2014
DOI: 10.1038/NCOMMS5339
Publisher: Springer Science and Business Media LLC
Date: 11-02-2010
DOI: 10.1038/LEU.2010.9
Abstract: SHIP-1 (SH2 (Src homology 2)-containing inositol 5'-phosphatase-1) functions as a negative regulator of immune responses by hydrolyzing phosphatidylinositol-3,4,5-triphosphate generated by phosphoinositide-3 (PI 3)-kinase activity. As a result, SHIP-1 deficiency in mice results in myeloproliferation and B-cell lymphoma. On the other hand, SHIP-1-deficient mice have a reduced T-cell population, but the underlying mechanisms are unknown. In this work, we hypothesized that SHIP-1 plays anti-apoptotic functions in T cells upon stimulation of the death receptor CD95/APO-1/Fas. Using primary T cells from SHIP-1(-/-) mice and T leukemic cell lines, we report that SHIP-1 is a potent inhibitor of CD95-induced death. We observed that a small fraction of the SHIP-1 pool is localized to the endoplasmic reticulum (ER), in which it promotes CD95 glycosylation. This post-translational modification requires an intact SH2 domain of SHIP-1, but is independent of its phosphatase activity. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired death-inducing signaling complex (DISC) formation and downstream apoptotic cascade. These results uncover an unanticipated inhibitory function for SHIP-1 and emphasize the role of glycosylation in the regulation of CD95 signaling in T cells. This work may also provide a new basis for therapeutic strategies using compounds inducing apoptosis through the CD95 pathway on SHIP-1-negative leukemic T cells.
Publisher: Public Library of Science (PLoS)
Date: 22-12-2009
Publisher: Elsevier BV
Date: 02-2010
Publisher: Springer Science and Business Media LLC
Date: 18-04-2008
Publisher: Springer Science and Business Media LLC
Date: 10-10-2022
DOI: 10.1038/S41598-022-20608-7
Abstract: Human cervicovaginal fluid (CVF) is a complex, functionally important and glycan rich biological fluid, fundamental in mediating physiological events associated with reproductive health. Using a comprehensive glycomic strategy we reveal an extremely rich and complex N-glycome in CVF of pregnant and non-pregnant women, abundant in paucimannose and high mannose glycans, complex glycans with 2–4 N-Acetyllactosamine (LacNAc) antennae, and Poly-LacNAc glycans decorated with fucosylation and sialylation. N-glycosylation profiles were observed to differ in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome. Compared to CVF from women experiencing term birth, CVF from women who subsequently experienced preterm birth showed lower sialylation, which correlated to the presence of a erse microbiome, and higher fucosylation, which correlated positively to pro-inflammatory cytokine concentration. This study is the first step towards better understanding the role of cervicovaginal glycans in reproductive health, their contribution to the mechanism of microbial driven preterm birth, and their potential for preventative therapy.
Publisher: The Royal Society
Date: 15-02-2019
Abstract: The lectin Helix pomatia agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum s les from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary N- linked glycans on IgA1 from BCa patients ( p 0.0001: non-core fucosylated p = 0.0345: core fucosylated) and increased asialo-Thomsen–Friedenreich antigen (TF) and disialo-TF antigens in the O- linked glycan preparations from IgA1 of cancer patients compared with healthy control in iduals. An increase in Sambucus nigra binding was observed, suggestive of increased α 2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases ( χ 2 13.359 n = 114 p = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue s les showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication.
Publisher: Oxford University Press (OUP)
Date: 29-01-2007
Abstract: The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single s le. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a s le and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Le(x) epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.
Publisher: Elsevier BV
Date: 03-1993
Publisher: Oxford University Press (OUP)
Date: 23-11-2010
Publisher: Springer Science and Business Media LLC
Date: 12-2013
Abstract: With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans -expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection.
Publisher: F1000 Research Ltd
Date: 06-2021
Publisher: Elsevier BV
Date: 12-2018
Publisher: Oxford University Press (OUP)
Date: 11-01-2012
Publisher: Elsevier BV
Date: 10-2006
Publisher: Elsevier BV
Date: 05-2013
Publisher: Springer Science and Business Media LLC
Date: 24-03-2011
Abstract: The Lewis x trisaccharide, also referred to as the CD15 antigen, is a diagnostic marker used to distinguish Hodgkin's lymphoma from other lymphocytic cancers. However, the role of such fucosylated structures remains poorly understood, in part because carriers of Lewis x structures on Hodgkin's Reed-Sternberg cells have not been identified. GalMBP, an engineered carbohydrate-recognition protein that binds selectively to oligosaccharides with paired terminal galactose and fucose residues, has been used in conjunction with proteomic and glycomic analysis to identify glycoprotein carriers of Lewis x and related glycan structures in multiple Hodgkin's Reed-Sternberg cell lines. Multiple glycoproteins that bind to GalMBP and carry CD15/Lewis x have been identified in a panel of six Reed-Sternberg cell lines. The most commonly identified Lewis x -bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were detected in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules on the lymphomas carry this characteristic glycan epitope. In addition, the Hodgkin's Reed-Sternberg cell lines can be grouped into subsets based on the presence or absence of less common Lewis x -bearing glycoproteins. CD98 and intercellular adhesion molecule-1 are major carriers of CD15/Lewis x on Reed-Sternberg cells. Binding of DC-SIGN and other glycan-specific receptors to the Lewis x epitopes on CD98 and intercellular adhesion molecule-1 may facilitate interaction of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes.
Publisher: Oxford University Press (OUP)
Date: 06-10-2015
Publisher: Elsevier BV
Date: 05-2006
Publisher: Elsevier BV
Date: 04-2013
Publisher: Springer Berlin Heidelberg
Date: 2001
Publisher: American Society of Hematology
Date: 29-11-2018
DOI: 10.1182/BLOOD-2018-99-117290
Abstract: Heterozygosity for Hemoglobin (Hb) S, sickle cell trait (SCT), affects over 40 million people and confers resistance to severe infection by Plasmodium falciparum. Homozygosity for HbS, or compound heterozygosity with certain other alleles of Hb, affects over 4 million in iduals and causes sickle cell disease (SCD). Hemolytic anaemia is a prominent feature of SCD and is mainly extravascular, mediated by hepatic and splenic macrophages. No ligands for this process have been identified. As many macrophage phagocytic receptors recognise carbohydrates, we surveyed surface glycan expression by sickle cells using a panel of 8 lectins and flow cytometry. Most glycans were similar to those of healthy red blood cells (RBC), except much higher expression of terminal mannose. We investigated the structural basis for these residues using glycomic mass spectroscopy, which showed them to be N-linked high (Man5-9GlcNAc2) mannoses, a surprising conclusion as these are usually intermediates in the formation of complex glycans and not displayed on cell surfaces. High resolution microscopy revealed the mannose residues to be carried in discrete microdomains on the surfaces of sickle cells. These structures were absent on the surfaces of healthy RBC, instead being present in the membrane skeleton under the cell surface. Lectin blots and immunoprecipitation showed the mannoses to co-migrate predominantly with spectrin. We showed these mannose-bearing structures were able to stimulate phagocytosis of RBC by using a peripheral blood derived macrophage uptake assay. Sickle RBC were taken up at high rates compared to healthy RBC and this could be inhibited by congeners of mannose. We identified the importance of a cognate ligand (CD206: the mannose receptor) using blocking antibodies and knockdown of CD206 expression using siRNA. The in vivo and pathogenic relevance of mannose exposure was investigated by taking advantage of the heterogeneity of hemolysis in SCD. RBC with SCD (n=94), SCT (n=57) and healthy in iduals (n=54) were assayed for mannose exposure by flow cytometry. SCT and healthy RBC showed no mannose exposure but high levels were found on HbSS RBC (p .0001). Co-incident inheritance of HbSS with higher HbF values and alleles encoding alpha-thalassaemia resulted in lower surface mannose values. Overall, markers of hemolysis (RBC count, haemoglobin, reticulocyte count) correlated well with mannose exposure (Spearman correlation coefficients -0.68, -0.40, 0.37 p=0.0001, 0.0032, 0.0063 respectively). Plasma LDH is a marker of intravascular hemolysis and correlated with overall hemolysis within SCD (r=-0.25, p=0.016), but not mannose exposure (r=0.14, p=0.19). Thus mannose exposure correlated only with extravascular hemolysis. Identification of a ligand pair mediating rapid clearance of sickle cells raised the possibility that they also mediate enhanced clearance of SCT RBC infected by malarial parasites. Indeed, P. falciparum cultures induced mannose expression at the pigmented trophozoite and schizont stages in infected HbAA RBCs, at levels corresponding to mild hemolysis in SCD. Mannose expression in infected HbAS RBCs was even higher, with levels corresponding to severe hemolysis in SCD. Infection with P. falciparum and selection for HbS arose only recently in human evolution, raising the question of what the physiological triggers for this mechanism are. Infection with malarial parasites causes oxidative stress. We therefore subjected healthy RBC to copper sulphate, which resulted in surface mannose exposure as well as uptake by macrophages. Oxidized SCT RBC displayed more mannose than oxidized healthy RBC. Thus, we have identified a new cell surface 'eat me' signalling mechanism that allows inspecting macrophages to engage with the rigid membrane skeleton and phagocytose the mannose displaying cell. The mechanism is stimulated by HbS: when present in high concentrations, the mechanism is activated constitutively, resulting in sickle cell anaemia. Heterozygosity for HbS is insufficient by itself to trigger mannose exposure. However, the mechanism is primed so that oxidative stress associated with infection by P. falciparum causes greater mannose display, increased parasitized cell clearance and protection against severe malaria. These findings should allow the design of inhibitors of sickle cell haemolysis and inducers of protection against malaria. Cao: University of Aberdeen: Patents & Royalties. Barker:University of Aberdeen: Patents & Royalties. Vickers:University of Aberdeen: Patents & Royalties GSK: Equity Ownership.
Publisher: Oxford University Press (OUP)
Date: 14-04-2010
Publisher: Oxford University Press (OUP)
Date: 08-03-2011
Publisher: Elsevier BV
Date: 05-2021
Publisher: Springer Science and Business Media LLC
Date: 17-08-2018
DOI: 10.1038/S41467-018-05770-9
Abstract: Leukocytes are coated with a layer of heterogeneous carbohydrates (glycans) that modulate immune function, in part by governing specific interactions with glycan-binding proteins (lectins). Although nearly all membrane proteins bear glycans, the identity and function of most of these sugars on leukocytes remain unexplored. Here, we characterize the N-glycan repertoire (N-glycome) of human tonsillar B cells. We observe that naive and memory B cells express an N-glycan repertoire conferring strong binding to the immunoregulatory lectin galectin-9 (Gal-9). Germinal center B cells, by contrast, show sharply diminished binding to Gal-9 due to upregulation of I-branched N-glycans, catalyzed by the β1,6- N -acetylglucosaminyltransferase GCNT2. Functionally, we find that Gal-9 is autologously produced by naive B cells, binds CD45, suppresses calcium signaling via a Lyn-CD22-SHP-1 dependent mechanism, and blunts B cell activation. Thus, our findings suggest Gal-9 intrinsically regulates B cell activation and may differentially modulate BCR signaling at steady state and within germinal centers.
Publisher: Oxford University Press (OUP)
Date: 03-09-2020
Abstract: The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.
Publisher: Elsevier BV
Date: 03-2005
Publisher: Informa UK Limited
Date: 07-2009
DOI: 10.1128/MCB.00204-09
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.INTIMP.2017.01.020
Abstract: Glycogen storage disease type Ib (GSD-Ib) is characterized by impaired glucose homeostasis, neutropenia and neutrophil dysfunction. Mass spectrometric glycomic profiling of GSD-Ib neutrophils showed severely truncated N-glycans, lacking galactose. Experiments indicated the hypoglycosylation of the electron transporting subunit of NADPH oxidase, which is crucial for the defense against bacterial infections. In phosphoglucomutase 1 (PGM1) deficiency, an inherited disorder with an enzymatic defect just one metabolic step ahead, hypogalactosylation can be successfully treated by dietary galactose. We hypothesized the same pathomechanism in GSD-Ib and started a therapeutic trial with oral galactose and uridine. The aim was to improve neutrophil dysfunction through the correction of hypoglycosylation in neutrophils. The GSD-Ib patient was treated for 29weeks. Monitoring included glycomics analysis of the patient's neutrophils and neutrophil function tests including respiratory burst activity, phagocytosis and migration. Although no substantial restoration of neutrophil glycosylation was found, there was partial improvement of respiratory burst activity.
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.VACCINE.2010.08.058
Abstract: Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 11-02-2005
Abstract: The development of pest resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins used in transgenic and organic farming. Here, we demonstrate that (i) the major mechanism for Bt toxin resistance in Caenorhabditis elegans entails a loss of glycolipid carbohydrates (ii) Bt toxin directly and specifically binds glycolipids and (iii) this binding is carbohydrate-dependent and relevant for toxin action in vivo. These carbohydrates contain the arthroseries core conserved in insects and nematodes but lacking in vertebrates. We present evidence that insect glycolipids are also receptors for Bt toxin.
Publisher: Oxford University Press (OUP)
Date: 27-10-2007
Abstract: Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to alpha2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy.
Publisher: Wiley
Date: 26-08-2008
DOI: 10.1038/ICB.2008.54
Abstract: The outermost layer of all immune cells, the glycocalyx, is composed of a complex mixture of glycoproteins, glycolipids and lectins, which specifically recognize particular glycan epitopes. As the glycocalyx is the cell's primary interface with the external environment many biologically significant events can be attributed to glycan recognition. For this reason the rapidly expanding glycomics field is being increasingly recognized as an important component in our quest to better understand the functioning of the immune system. In this review, we highlight the current status of immune cell glycomics, with particular attention being paid to T- and B-lymphocytes and dendritic cells. We also describe the strategies and methodologies used to define immune cell glycomes.
Publisher: American Chemical Society (ACS)
Date: 02-10-2009
DOI: 10.1021/PR9001756
Abstract: A recent analysis of the human sperm N-glycome confirmed the expression of biantennary bisecting type N-glycans and terminal Lewis(x)/Lewis(y) sequences previously implicated in the suppression of the innate and adaptive immune responses, respectively. In this study, glycomic analysis of seminal plasma glycoproteins derived from four fertile men was carried out to determine if the same sequences were expressed on the N- and O-glycome of human seminal plasma glycoproteins. Three major families of N-glycans were detected: (i) high mannose glycans (Man(5-7)GlcNAc(2)) (ii) bi-, tri-, and tetraantennary core-fucosylated complex type N-glycans with antennae terminated with Lewis(x) and/or Lewis(y) sequences and (iii) bi-, tri-, and tetraantennary core-fucosylated complex type N-glycans with antennae capped with sialic acid. Analysis of the O-glycans revealed Core 1 and Core 2 type structures that are also fucosylated or sialylated or a combination of both. The same high mannose and polyfucosylated N-glycans associated with sperm are also present in seminal plasma. Bisecting type N-glycan expression is greatly decreased compared to sperm, while sialylated glycans are abundant in some in iduals and minor in others. In summary, the glycosylation profile of seminal plasma glycoproteins is consistent with the modulation of the adaptive but not the innate arm of the human immune response.
Publisher: Wiley
Date: 07-11-2012
Abstract: Human native milk lactoferrin (LF) and recombinant forms of lactoferrin (rLF) are available with identical aa sequences, but different glycosylation patterns. Native lactoferrin (NLF) possesses the intrinsic ability to stimulate vigorous IgG and IgE antibody responses in BALB/c mice, whereas recombinant forms (Aspergillus or rice) are 40-fold less immunogenic and 200-fold less allergenic. Such differences are independent of endotoxin or iron content and the glycans do not contribute to epitope formation. A complex glycoprofile is observed for NLF, including sialic acid, fucose, mannose, and Lewis (Le)(x) structures, whereas both rLF species display a simpler glycoprofile rich in mannose. Although Le(x) type sugars play a Th2-type adjuvant role, endogenous expression of Le(x) on NLF did not completely account for the more vigorous IgE responses it provoked. Furthermore, coadminstration of rLF downregulated IgE and upregulated IgG2a antibody responses provoked by NLF, but was without effect on responses to unrelated peanut and chicken egg allergens. These results suggest glycans on rLF impact the induction phase to selectively inhibit IgE responses and that differential glycosylation patterns may impact on antigen uptake, processing and/or presentation, and the balance between Th1 and Th2 responses.
Publisher: American Society of Hematology
Date: 29-11-2018
DOI: 10.1182/BLOOD-2018-99-114568
Abstract: Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet count (≤100x109/L) due to platelet destruction and insufficient platelet production. There is a wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments. It has been proposed that certain autoimmune diseases might be caused by changes in glycosylation of cell surface proteins which induce the immune system to recognize cells as "non-self". The aim of this work was to evaluate whether glycosylation might be involved in ITP ethiopathogenesis. So, in this preliminary study we aimed to evaluate the glycosylation pattern of platelets from ITP patients, its relationship with platelet features and with the response to treatments. Methods: Seventy eight ITP patients were recruited: 32 without treatment (UT-ITP) for at least six months, 35 responders to thrombopoietin receptor agonists (TPO-RA, 62.8% with eltrombopag, 27.2 % with romiplostim), and 11 refractory (r-ITP). Eighty one healthy controls were also included. Human peripheral blood s les were collected in 3.8% sodium citrate. Platelet activation was determined by flow cytometry (FCM) through binding of FITC-PAC1 (a mAb that recognizes the activated conformation of fibrinogen receptor) after activation with 100 mM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland). Apoptosis was evaluated by measurement of active caspase-3, -8 or -9 by FCM with a specific kit (Millipore, Madrid, Spain). Platelet surface exposure of lectins was analyzed with 1 µg/ml FITC-conjugated Wheat germ agglutinin lectin (WGA) or 1 µg/ml FITC-conjugated Erythrina cristagalli lectin (ECA). WGA binds to sialic acid and N-acetylglucosaminyl residues and ECA is a galactose specific legume lectin. Glycoprotein derived glycans from a healthy control and from a r-ITP patient were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion whilst O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-MS. Comparisons of quantitative variables were made with SPSS.22 software. Results: Platelet count (x109/L) was 269±55 in controls, 141±126 in UT-ITP, 111±85 in TPO-RA and 8±5 in r-ITP. ITP patients with the lowest platelet count showed the most pronounced binding of WGA and ECA (Table 1). Moreover, binding of these lectins showed a mild positive correlation with caspase activities (Table 1). No relationship was observed between platelet ability to be activated and glycosylation features. When lectins' binding was studied stratifying patients according to their response to therapeutic treatments we observed that platelets from all ITP patients bound more ECA and WGA (Figure 1). Despite no significant differences were observed between ITP groups, r-ITP seemed to bind more WGA. To increase our knowledge about protein glycosylation of platelet proteins in ITP patients we performed glycomic analyses of platelets from one healthy control and from one r-ITP patient. It was demonstrated that bi-, tri- and tetra-antennary complex type N-glycans are the dominant class of N-glycans with also some high mannose structures. Analysis of sialylated N-glycan, via digestion with sialidase, revealed lower levels of sialylation in r-ITP patient platelets compared to healthy control platelets. Platelet O-glycan data revealed the presence of mucin-type core-1 and core-2 structures. Conclusion: Glycomic analysis demonstrates that human platelet glycoproteins are modified with a complex array of both N- and O-linked glycans. Preliminary data indicates that there could be changes in glycosylation associated with immune thrombocytopenia. These changes in platelet protein glycosylation might be involved in an enhancement of apoptosis in platelets from ITP patients. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Álvarez-Roman: Shire: Consultancy NovoNordisk: Consultancy SOBI: Consultancy. Jimenez-Yuste:Bayer: Consultancy, Research Funding Sobi: Consultancy, Research Funding Pfizer: Consultancy, Research Funding Roche: Consultancy, Research Funding Grifols: Consultancy, Research Funding Octapharma: Consultancy, Research Funding CSL Behring: Consultancy NovoNordisk: Consultancy, Research Funding Shire: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.
Publisher: Cold Spring Harbor Laboratory
Date: 27-11-2020
DOI: 10.1101/2020.11.26.399402
Abstract: In both sickle cell disease (SCD) and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man 5-9 GlcNAc 2 ), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. Extravascular haemolysis in SCD correlates with high mannose glycan levels on RBCs. Infection of RBCs with Plasmodium falciparum expose surface mannose N-glycans on healthy RBCs, which occurred at significantly higher levels on RBCs from subjects with sickle cell trait compared to those lacking haemoglobin S. The glycans were associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans, a novel response to cellular stress, is the first molecular mechanism common to both the pathogenesis of SCD and resistance to severe malaria in sickle cell trait.
Publisher: Elsevier BV
Date: 09-2023
Publisher: Elsevier BV
Date: 2007
Publisher: Elsevier BV
Date: 05-2009
Publisher: Elsevier BV
Date: 10-2005
Publisher: Springer Science and Business Media LLC
Date: 20-04-2021
DOI: 10.1038/S41467-021-22365-Z
Abstract: In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation.
Publisher: Elsevier
Date: 2023
Publisher: Elsevier BV
Date: 10-2010
Publisher: Elsevier BV
Date: 09-2006
Publisher: Hindawi Limited
Date: 2010
DOI: 10.1155/2010/148178
Abstract: Recent years have witnessed a rapid growth in the number and ersity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i) oligosaccharyltransferase (OST)-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria (ii) stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain (iii) OST-mediated O-glycosylation which appears to be characteristic of bacteria and (iv) stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER eriplasmic/cytoplasmic membranes, and transferring “ en bloc ” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on erse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.
Publisher: Elsevier BV
Date: 09-2023
Publisher: American Society of Hematology
Date: 15-11-2013
DOI: 10.1182/BLOOD.V122.21.439.439
Abstract: Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophil granulocytes. Mutations in ELANE, GFI1, HAX1, G6PC3, WAS, and VPS45 are known to cause SCN. We here describe a new monogenetic SCN variant with biallelic mutations in the gene encoding Jagunal homolog 1 (JAGN1). We studied an index family from Northern Africa with a total of 5 children suffering from SCN. An Affymetrix SNP array-based genetic linkage analysis was performed and identified a single interval of perfect segregation with highly significant multi-marker LOD scores of at least 4.5spanning approximately 1.5Mbp from 9.52Mb to 11.04Mb on chromosome 3 of NCBI’s human genome build 36.3. This interval contained a total of 30 genes, including JAGN1 which encodes an ER-resident protein. Sanger sequencing revealed a homozygous mutation c.3G A in exon 1 of the JAGN1 gene this mutation leads to disruption of the defined start of translation. Systematic analysis of a cohort of 90 SCN patients identified 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients, thus accounting for approximately 10% of SCN patients. The clinical phenotype was variable and included failure to thrive, developmental delay and bone skeletal abnormalities. The only consistent finding in all JAGN1-deficient patients was SCN and partial or complete refractoriness to therapy with rh-GCSF. JAGN1 is the human ortholog of a gene originally identified in Drosophila melanogaster. Jagunal-deficient oocytes are characterized by defective ER reorganization and aberrant membrane trafficking during vitellogenesis. We found that JAGN1-mutant human granulocytes showed aberrantly enlarged ER structures and paucity of secretory vesicles. We hypothesized that that ER aberrations may be associated with defective N-glycosylation of multiple proteins in neutrophil granulocytes and found that JAGN1-mutant neutrophil granulocytes exhibited anomalous N-glycomic profiles characterized by a marked reduction in fucosylation of all their multi-antennary glycans. JAGN1-deficient neutrophil granulocytes showed increased apoptosis in response to TNFa and staurosporine, likely accounting for the lack of mature neutrophils in these patients. Additional studies in JAGN1-knockdown cells indicate that JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. Global proteomic analysis of the JAGN1-interactome identified a limited number of interaction partners including members of the Coat Protein I (COPI) complex (COPA, COPB2, and COPG2) which suggest a role for JAGN1 in vesicular trafficking from Golgi to ER. Taken together, JAGN1 emerges as a hitherto unrecognized factor necessary in differentiation and survival of neutrophil granulocytes and a novel gene implicated in SCN. No relevant conflicts of interest to declare.
Publisher: Elsevier BV
Date: 02-2007
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.SBI.2006.08.006
Abstract: Mass spectrometry has been at the forefront of glycobiological analysis for more than two decades. Recent advances in technology, coupled with increased global appreciation of the importance of the glycome in solving biological problems, have laid the foundations for an unprecedented increase in output. Under the auspices of major international consortia, significant progress is being made towards the development of integrated databases of specific cell and tissue N- and O-glycan profiles. Tools for the automation of both s le handling and data analysis are enabling a more concerted approach to glycan structural analysis. When combined with complementary transcriptomics and bioinformatics analyses, these tools will facilitate a new systems glycobiology approach to research.
Publisher: American Chemical Society (ACS)
Date: 11-07-2012
DOI: 10.1021/BC200624A
Abstract: The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide rotein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.
Publisher: Wiley
Date: 2011
DOI: 10.1002/BAB.7
Abstract: We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.
Publisher: Elsevier
Date: 2006
Publisher: Wiley
Date: 10-2008
Publisher: American Chemical Society (ACS)
Date: 11-09-2013
DOI: 10.1021/CB400486T
Abstract: Cell-cell communications, cell-matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan-receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan-receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan-receptor interactions in the regeneration of H. magnipapillata.
Publisher: Public Library of Science (PLoS)
Date: 25-08-2011
Publisher: Oxford University Press (OUP)
Date: 11-05-2009
Publisher: Elsevier BV
Date: 10-2016
Publisher: Springer Science and Business Media LLC
Date: 10-06-2012
DOI: 10.1038/NCHEMBIO.999
Publisher: Wiley
Date: 12-04-2022
DOI: 10.1111/BJH.18164
Abstract: Red blood cells (RBCs) lose plasma membrane in the spleen as they age, but the cells and molecules involved are yet to be identified. Sickle cell disease and infection by Plasmodium falciparum cause oxidative stress that induces aggregates of cross‐linked proteins with N‐linked high‐mannose glycans (HMGs). These glycans can be recognised by mannose‐binding lectins, including the mannose receptor (CD206), expressed on macrophages and specialised phagocytic endothelial cells in the spleen to mediate the extravascular haemolysis characteristic of these diseases. We postulated this system might also mediate removal of molecules and membrane in healthy in iduals. Surface expression of HMGs on RBCs from patients who had previously undergone splenectomy was therefore assessed: high levels were indeed observable as large membrane aggregates. Glycomic analysis by mass spectrometry identified a mixture of Man 5‐9 GlcNAc 2 structures. HMG levels correlated well with manual pit counts ( r = 0.75–0.85). To assess further whether HMGs might act as a splenic reticuloendothelial function test, we measured levels on RBCs from patients with potential functional hyposplenism, some of whom exhibited high levels that may indicate risk of complications.
Publisher: Oxford University Press (OUP)
Date: 06-2006
Publisher: Elsevier BV
Date: 09-2006
Publisher: American Thoracic Society
Date: 05-2005
Publisher: Oxford University Press (OUP)
Date: 08-2023
DOI: 10.1093/PNASNEXUS/PGAD247
Abstract: Placental abnormalities cause impaired fetal growth and poor pregnancy outcome (e.g. preecl sia [PE]) with long-lasting consequences for the mother and offspring. The molecular dialogue between the maternal niche and the developing placenta is critical for the function of this organ. Galectin-1 (gal-1), a highly expressed glycan-binding protein at the maternal–fetal interface, orchestrates the maternal adaptation to pregnancy and placenta development. Down-regulation or deficiency of gal-1 during pregnancy is associated with the development of PE however, the maternal- and placental-derived gal-1 contributions to the disease onset are largely unknown. We demonstrate that lack of gal-1 imposes a risk for PE development in a niche-specific manner, and this is accompanied by a placental dysfunction highly influenced by the absence of maternal-derived gal-1. Notably, differential placental glycosylation through the Sda-capped N-glycans dominates the invasive trophoblast capacity triggered by maternal-derived gal-1. Our findings show that gal-1 derived from the maternal niche is essential for healthy placenta development and indicate that impairment of the gal-1 signaling pathway within the maternal niche could be a molecular cause for maternal cardiovascular maladaptation during pregnancy.
Publisher: Elsevier
Date: 2010
Publisher: American Society of Hematology
Date: 05-11-2021
DOI: 10.1182/BLOOD-2021-150771
Abstract: Introduction: Platelet glycoproteins are key contributors to platelet function but their glycans structure is unclear. Alterations in glycan composition have been reported to impact platelet clearance under physiological conditions and in the disease mechanism of immune thrombocytopenia (ITP). Therefore, this study sought to characterize glycan structures in human platelets from healthy control in iduals and ITP patients using mass spectrometry (MS)-based glycomics approach, andto compare their glycomic profiles to facilitate understanding of glycan alterations in ITP. Methods: Glycan residues on platelet surface were determined by flow cytometry. Platelet lysates (1×10 8 platelets) from 4 healthy controls and from 4 ITP patients with a clear anomalous glycosylation pattern were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion and subsequently purified with a Sep-Pak C18 reverse phase cartridge. O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-TOF MS to obtain an initial carbohydrate profile. Selected glycan molecular ion species were analyzed by MALDI-TOF-TOF MS/MS before and after digesting with exoglycosidases. Results: Glycans present in platelets from healthy controls and from ITP patients were largely consistent. The MS spectra for N-glycans showed a mixture of high mannose glycans (m/z 1579.8, 1783.9, 1988.0, 2192.1 and 2396.2) complex glycans (m/z 2966.5, 3776.9 and 4587.4) and bisected or truncated glycans (m/z 3211.6 and 4022.1). The spectra showed the presence of bi-, tri- and tetra-antennary complex glycans, which varied in their level of sialylation.Figure 1 shows the relative abundance ratio within eight families of core glycan structures. Platelets from ITP patients showed a consistent increase in desialylated structures such as Hex 5HexNAc 4Fuc 1. In addition to different amounts of attached sialic acid, varying levels of fucosylation were observed ranging from the addition of a single core Fuc (m/z 2244.1), to the addition of up to three Fuc residues (m/z 3402.7). Collision-activated decomposition (CAD) MALDI-TOF/TOF analysis was performed to generate fragment ions from molecular ions detected in MALDI-TOF profiling for detailed sequencing of platelet N-glycans. This analysis suggested the presence of three isoforms: (i) sialylated tetra-antennary structure with core fucosylation and one Lewis x/a antenna (ii) sialylated tetra-antennary structure with one Lewis y/b antenna (ii) sialylated, core-fucosylated tri-antennary structure with one LacNAc extension and one Lewis x/a antenna. Sialidase S digestion was used to highlight the extent of desialylation in the presence of sialidase. Noteworthy, the ratio of non-sialylated bi-and tri-antennary N-glycans in ITP patients were higher than that in controls. This enzymatic digestion confirms the presence of α2,3-linked Neu5Ac on platelet glycans. Remaining sialylated structures may possess α2,6-linked Neu5Ac. O-glycan profiles obtained showed the predominance of core 1 and core 2 structures (Figure 2). The two most dominant glycan structures in core 1 were sialyl T antigen (GalNAc 1Gal 1NeuAc 1 m/z 895.5) and disialyl T antigen (GalNAc 1Gal 1NeuAc 2 m/z 1256.7), being the latter less abundant in ITP patients. The core 2 structure was modified by the addition of fucose and/or sialic acid residues (m/z 1157.7, 1344.8, 1518.9 and 1706.0). Conclusion: N- and O-glycan structures in human platelets were characterized by MALDI-TOF MS profiling to reveal interesting structural features including the presence of sialylLewis x/a epitope, Lewis x/a epitope, Lewis y/b epitope and LacNAc extensions in complex type N-glycans. Presence of terminal sialic acid and sialylLewis x/a on platelet N-glycan antenna also suggest their potentialfunction as ligands for siglecs that are associated with cell signaling functions. Siglec-1 and -2 have been suggested to have potential roles in ethiopathogenesis of autoimmune diseases. Desialylation observed in glycans of platelets from ITP patients, might trigger immune system activation in these patients. This research was funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Butta: Roche: Speakers Bureau Takeda: Research Funding, Speakers Bureau CSL-Behring: Research Funding Novo-Nordisk: Speakers Bureau. Canales: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau Karyopharm: Consultancy, Honoraria Takeda: Consultancy, Honoraria, Speakers Bureau iQone: Honoraria Sandoz: Honoraria, Speakers Bureau Incyte: Consultancy Janssen: Consultancy, Honoraria, Speakers Bureau Gilead/Kite: Consultancy, Honoraria Novartis: Consultancy, Honoraria Sanofi: Consultancy Eusa Pharma: Consultancy, Honoraria Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Jiménez-Yuste: Pfizer: Consultancy, Honoraria, Research Funding F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding BioMarin: Consultancy Sobi: Consultancy, Honoraria, Research Funding NovoNordisk: Consultancy, Honoraria, Research Funding Octapharma: Consultancy, Honoraria, Research Funding Sanofi: Consultancy, Honoraria, Research Funding Takeda: Consultancy, Honoraria, Research Funding Bayer: Consultancy, Honoraria, Research Funding CSL Behring: Consultancy, Honoraria, Research Funding Grifols: Consultancy, Honoraria, Research Funding. Alvarez Román: Octapharma: Consultancy, Honoraria, Research Funding Biomarin: Consultancy, Honoraria, Research Funding Novartis: Consultancy, Honoraria, Research Funding Amgen: Consultancy, Honoraria, Research Funding Pfizer: Consultancy, Honoraria, Research Funding Bayer: Consultancy, Honoraria, Research Funding CSL-Behring: Consultancy, Honoraria, Research Funding Grifols: Consultancy, Honoraria, Research Funding Novo-Nordisk: Consultancy, Honoraria, Research Funding Sobi: Consultancy, Honoraria, Research Funding Takeda: Consultancy, Honoraria, Research Funding.
Publisher: Wiley
Date: 07-04-2005
DOI: 10.1016/J.FEBSLET.2005.03.071
Abstract: Fibroblasts are a erse cell type and display clear topographic differentiation and positional memory. In a screen for fibroblast specific markers we have characterized four monoclonal antibodies to endosialin (TEM1/CD248). Previous studies have reported that endosialin is a tumour endothelium marker and is localized intracellularly. We demonstrate conclusively that endosialin is a cell surface glycoprotein and is predominantly expressed by fibroblasts and a subset of pericytes associated with tumour vessels but not by tumour endothelium. These novel antibodies will facilitate the isolation and classification of fibroblast and pericyte lineages as well as the further functional analysis of endosialin.
Publisher: American Society for Microbiology
Date: 07-2008
DOI: 10.1128/JVI.02731-07
Abstract: The Ebola virus nucleoprotein (NP) is an essential component of the nucleocapsid, required for filovirus particle formation and replication. Together with virion protein 35 (VP35) and VP24, this gene product gives rise to the filamentous nucleocapsid within transfected cells. Ebola virus NP migrates aberrantly, with an apparent molecular mass of 115 kDa, although it is predicted to encode an ∼85-kDa protein. In this report, we show that two domains of this protein determine this aberrant migration and that this region mediates its incorporation into virions. These regions, amino acids 439 to 492 and amino acids 589 to 739, alter the mobility of Ebola virus NP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by 5 and 15 kDa, respectively, and confer similar effects on a heterologous protein, LacZ, in a position-independent fashion. Furthermore, when coexpressed with VP40, VP35, and VP24, this region mediated incorporation of NP into released viruslike particles. When fused to chimeric paramyxovirus NPs derived from measles or respiratory syncytial virus, this domain directed these proteins into the viruslike particle. The COOH-terminal NP domain comprises a conserved highly acidic region of NP with predicted disorder, distinguishing Ebola virus NPs from paramyxovirus NPs. The acidic character of this domain is likely responsible for its aberrant biochemical properties. These findings demonstrate that this region is essential for the assembly of the filamentous nucleocapsids that give rise to filoviruses.
Publisher: Wiley
Date: 27-03-2007
DOI: 10.1111/J.1742-4658.2007.05746.X
Abstract: The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.
Publisher: Elsevier BV
Date: 12-2005
DOI: 10.1016/J.YGYNO.2005.07.030
Abstract: CA125 expresses specific oligosaccharides that can inhibit the cytotoxicity of human natural killer (NK) cells. The current study was undertaken to determine the ability of CA125 to modulate NK cell-mediated cytotoxicity. CA125 was isolated from OVCAR-3 cells and its purity was determined by ELISA and ultra-sensitive mass spectrometric analysis. Peripheral blood-derived NK were treated with CA125 and standard cytotoxicity assays were performed using 51Cr-labeled K562 cells as targets. The expression of cell surface and intracellular markers on NK cells was determined by either flow cytometry or Western blot analysis. NK cells incubated with CA125 for 72 h exhibited a 50-70% decrease in the lysis of K562 targets. Incubation with CA125 for 4 h and 24 h had no effect on NK-mediated cytolysis. Inhibition of NK function was observed at CA125 concentrations (10,000-100,000 U/ml) that are expected to be significantly lower than those observed in the tumor microenvironment. Co-stimulation with IL-2 did not abrogate the NK inhibitory response of CA125. CA125 did not reduce proliferation or induce apoptosis of NK cells and alter the expression of p56lck, phospholipase Cgamma1, ZAP70, or CD3zeta. CA125 did, however, induce major downregulation of CD16 and minor decrease in expression of CD94/NKG2A. Our ongoing research and recent work performed by other laboratories highlights the potential physiologic role of this mucin. Based on the data presented here, it is likely that the tumor-derived CA125 acts as a suppressor of the immune response that is directed against the ovarian tumors.
Publisher: American Chemical Society (ACS)
Date: 19-08-2004
DOI: 10.1021/TX049935+
Abstract: The covalent binding of 4-[(13)C]- and 5-[(13)C]-5-chloro-2-methylisothiazol-3-one (MCI) toward human serum albumin (HSA) was followed by (13)C and (1)H[(13)C] NMR spectroscopy. MCI was found to react with histidine through an addition-elimination at position 5, leading to stable substitution adducts, and with lysine to form open adducts of the thioamide or amide type. No other modification could be detected on either cysteine or tyrosine. In the presence of glutathione (GSH), we observed an increased covalent binding to lysine residues. This could be explained by the rapid reaction of GSH with MCI to form a chlorothioacyl intermediate very reactive toward primary amino groups of lysine residues. To further confirm these observations and map covalent binding sites, HSA s les modified by MCI with or without GSH were analyzed by matrix-assisted laser desorption/ionization mass spectrometry of tryptic digests and electrospray tandem mass spectrometry of modified peptides purified by reverse phase HPLC. About 80% of the HSA sequence was mapped, and several modified peptides were identified. When HSA was incubated with MCI without GSH, three peptides modified at histidine residues were characterized while when HSA was incubated in the presence of GSH, five peptides modified at histidine and lysine residues were identified. These experiments confirmed that modifications on lysine residues were of the amide and thioamide types. Observed modifications were in accordance with mass increases corresponding to structures identified by NMR, and an extra adduct corresponding to a double modification of His 338 was observed. Comparison of HSA-MCI and HSA-MCI-GSH s les confirmed that the presence of GSH increased the modification of lysine residues.
Publisher: Oxford University Press (OUP)
Date: 09-2016
Abstract: The minimum 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 s le 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 s le preparation. The s le preparation guidelines include all aspects of s le generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE s le preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.
Publisher: Elsevier BV
Date: 03-2005
DOI: 10.1016/J.CARRES.2004.12.016
Abstract: The bacterium Moraxella bovis is the causative agent of an economically important disease of cattle: Infectious Bovine Keratoconjunctivitis (IBK), otherwise known as pinkeye. Little is known regarding the structure of the carbohydrates produced by M. bovis. The structure of a capsular polysaccharide from M. bovis (strain Mb25) has been determined using NMR and MS analysis. From these data it is concluded that the polysaccharide is composed of the unmodified chondroitin disaccharide repeat unit.
Publisher: American Association for Cancer Research (AACR)
Date: 03-2014
DOI: 10.1158/2159-8290.CD-13-0287
Abstract: To interrogate the complex mechanisms involved in the later stages of cancer metastasis, we designed a functional in vivo RNA interference (RNAi) screen combined with next-generation sequencing. Using this approach, we identified the sialyltransferase ST6GalNAc2 as a novel breast cancer metastasis suppressor. Mechanistically, ST6GalNAc2 silencing alters the profile of O-glycans on the tumor cell surface, facilitating binding of the soluble lectin galectin-3. This then enhances tumor cell retention and emboli formation at metastatic sites leading to increased metastatic burden, events that can be completely blocked by galectin-3 inhibition. Critically, elevated ST6GALNAC2, but not galectin-3, expression in estrogen receptor–negative breast cancers significantly correlates with reduced frequency of metastatic events and improved survival. These data demonstrate that the prometastatic role of galectin-3 is regulated by its ability to bind to the tumor cell surface and highlight the potential of monitoring ST6GalNAc2 expression to stratify patients with breast cancer for treatment with galectin-3 inhibitors. Significance: RNAi screens have the potential to uncover novel mechanisms in metastasis but do not necessarily identify clinically relevant therapeutic targets. Our demonstration that the sialyltransferase ST6GalNAc2 acts as a metastasis suppressor by impairing binding of galectin-3 to the tumor cell surface offers the opportunity to identify patients with breast cancer suitable for treatment with clinically well-tolerated galectin-3 inhibitors. Cancer Discov 4(3) 304–17. ©2014 AACR. See related commentary by Ferrer and Reginato, p. 275 This article is highlighted in the In This Issue feature, p. 259
Publisher: Elsevier BV
Date: 02-2005
DOI: 10.1016/J.BBAGEN.2004.11.010
Abstract: The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.
Publisher: Elsevier BV
Date: 10-2014
Publisher: Elsevier BV
Date: 06-2011
Publisher: Oxford University Press (OUP)
Date: 13-04-2020
Abstract: Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.
Publisher: American Society for Microbiology
Date: 30-04-2019
Abstract: Advances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of general N- linked glycosylation in a major food poisoning bacterium, C ylobacter jejuni . The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in the C. jejuni proteome. Our quantitative proteomic results linked general protein N- glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterial N- glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria.
Publisher: Elsevier BV
Date: 03-2018
Publisher: Elsevier BV
Date: 06-2016
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-2018
Publisher: American Chemical Society (ACS)
Date: 03-2008
DOI: 10.1021/PR7008252
Abstract: Mass spectrometry is the main analytical technique currently used to address the challenges of glycomics as it offers unrivalled levels of sensitivity and the ability to handle complex mixtures of different glycan variations. Determination of glycan structures from analysis of MS data is a major bottleneck in high-throughput glycomics projects, and robust solutions to this problem are of critical importance. However, all the approaches currently available have inherent restrictions to the type of glycans they can identify, and none of them have proved to be a definitive tool for glycomics. GlycoWorkbench is a software tool developed by the EUROCarbDB initiative to assist the manual interpretation of MS data. The main task of GlycoWorkbench is to evaluate a set of structures proposed by the user by matching the corresponding theoretical list of fragment masses against the list of peaks derived from the spectrum. The tool provides an easy to use graphical interface, a comprehensive and increasing set of structural constituents, an exhaustive collection of fragmentation types, and a broad list of annotation options. The aim of GlycoWorkbench is to offer complete support for the routine interpretation of MS data. The software is available for download from: pplications/ms-tools.
Publisher: Wiley
Date: 11-11-2008
DOI: 10.1111/J.1471-4159.2008.05724.X
Abstract: While glycosyltransferases are restrictively expressed in invertebrate model organisms, little is known of their glycan end products. One such restrictively expressed glycoepitope was localized to sensory and epithelial cells of leech and Caenorhabditis elegans using the Lan3-2 monoclonal antibody. A biological function for the neural Lan3-2 epitope was previously determined in the leech. Here we report on the chemical structure of this mannosidic epitope harvested from whole Hirudo medicinalis. Crude glycans were liberated from glycoproteins by hydrazinolysis. Re-N-acetylated glycans were subjected to immunoaffinity purification. The affinity-purified glycans were fractioned by size chromatography into oligosaccharides and polysaccharides. Lan3-2 oligosaccharide structure was characterized by gas chromatography of alditol acetates, methylation analysis, 500 MHz 1H NMR spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem MS-MS of permethylated derivatives. The predominant components of the Lan3-2 oligosaccharide fraction were a series of linear beta-(1,4)-linked mannose polymers. The homologous expression of the Lan3-2 epitope in C. elegans will facilitate the exploration of its glycosylation pathway. Other invertebrates expressing the Lan3-2 epitope are Planaria dugesia, Capitella sp. I and Lumbriculus variegatus. The glycoepitope was not detected in the diploblastic animals Hydra littoralis and Aptaisia sp. or in deuterostomes.
Publisher: Oxford University Press (OUP)
Date: 12-01-2007
Abstract: Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the ergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same s les of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these s les. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.
Publisher: Oxford University Press (OUP)
Date: 16-12-2009
Publisher: Elsevier BV
Date: 08-2015
Publisher: Public Library of Science (PLoS)
Date: 23-08-2011
Publisher: Microbiology Society
Date: 08-2007
Publisher: American Society for Microbiology
Date: 07-2008
DOI: 10.1128/JVI.00187-08
Abstract: Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind α2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as in idual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.
Publisher: Wiley
Date: 17-10-2008
DOI: 10.1002/JMS.1514
Abstract: Like many other bacterial cell surfaces, the cell wall of Clostridium difficile is also encapsulated by a proteinaceous paracrystalline layer, the surface (S)-layer. In many bacterial species, the S-layer proteins (SLPs) have been shown to be glycosylated, whereas in other species glycosylation is absent. Unusually, the S-layer of C. difficile is composed of two distinct proteins, the high-molecular weight (HMW) and low-molecular-weight (LMW) SLPs. Previous investigations have reported that one or both of these SLPs are glycosylated, though no definitive study has been conducted. We have used a variety of mass spectrometric approaches to analyse SLPs from a number of strains of C. difficile for the presence of associated glycans. Analysis of intact SLPs by matrix assisted laser desorption/ionisation time of flight (MALDI-ToF) mass spectrometry demonstrated that the observed molecular masses matched the predicted masses of the LMW and HMW SLPs. Furthermore, analysis of Cyanogen bromide (CNBr) and tryptic peptides displayed no evidence of post-translational modification. In the first in-depth study of its kind, we unequivocally demonstrate that the S-layer proteins from the C. difficile strains investigated are not glycosylated.
Publisher: Elsevier BV
Date: 02-2007
Publisher: Elsevier BV
Date: 12-2016
Publisher: Oxford University Press (OUP)
Date: 05-2007
Publisher: Oxford University Press (OUP)
Date: 08-06-2010
Publisher: Elsevier BV
Date: 08-2008
DOI: 10.1016/J.TIV.2008.03.006
Abstract: A large proportion of allergic skin reactions are considered to be the result of skin exposure to small organic chemicals that possess the intrinsic ability to covalently modify skin proteins, either directly or following activation. In the absence of information about specific skin protein targets, studies of chemical modifications are limited to the use of model proteins. We have previously demonstrated that selected well known skin sensitizers (2,4-dinitro-1-chlorobenzene and phenyl salicylate) have the ability to covalently modify residues selectively on the model protein, human serum albumin. In the present work, we focus on the differences in covalent binding observed for two additional model proteins, human cytokeratin 14 and human cofilin, both constituent proteins of skin. Using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano LC-MS and -MS/MS strategies, the amino acid residues targeted by 2,4-dinitro-1-chlorobenzene on the two model proteins have been identified. In contrast, a structurally related non-sensitiser (2,4-dichloro-1-nitrobenzene) and a non-sensitising irritant (benzalkonium chloride) did not covalently modify the model proteins. Detailed examination of the results for the sensitizers indicate that reactive chemicals target nucleophilic amino acids residing in specific microenvironments of the 3D protein structure that are conducive to reactivity. This observation has important implications for the development of hapten-peptide binding assays. It is envisaged that the data from such assays will be integrated with outputs from other in vitro assays in the future to give a prediction of the sensitisation potential of novel chemicals.
Publisher: Proceedings of the National Academy of Sciences
Date: 09-02-2005
Abstract: C ylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli . In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N -glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.
Publisher: Elsevier BV
Date: 2012
Publisher: Oxford University Press (OUP)
Date: 05-11-2015
Publisher: Elsevier BV
Date: 09-2005
Publisher: Public Library of Science (PLoS)
Date: 27-01-2014
Publisher: Elsevier BV
Date: 09-2007
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2343-4_1
Abstract: The GlycoWorkbench software tool allows users to semiautomatically annotate glycomics MS and MS/MS spectra and MS glycoproteomics spectra. The GlycanBuilder software tool is embedded within GlycoWorkbench allowing users to draw glycan structures and export images of the drawn structures. This chapter demonstrates to users how to draw glycan structures within GlycoWorkbench using the GlycanBuilder software tool. This chapter also demonstrates how to use GlycoWorkbench to import MS and MS/MS glycomics spectra and use the cascading annotation feature to annotate both the MS and MS/MS spectra with a single command.
Publisher: Oxford University Press (OUP)
Date: 06-12-2014
Publisher: Elsevier BV
Date: 04-2006
DOI: 10.1016/J.BBAGEN.2005.12.013
Abstract: Defects in glycosylation are becoming increasingly associated with a range of human diseases. In some cases, the disease is caused by the glycosylation defect, whereas in others, the aberrant glycosylation may be a consequence of the disease. The implementation of highly sensitive and rapid mass spectrometric screening strategies for profiling the glycans present in model biological systems is revealing valuable insights into disease phenotypes. In addition, glycan screening is proving useful in the analysis of knock-out mice where it is possible to assess the role of glycosyltransferases and glycosidases and what function they have at the cellular and whole organism level. In this study, we analysed the effect of insulin on the glycosylation of 3T3-L1 cells and the effect of insulin resistance on glycosylation in a mouse model. Transcription profiling of 3T3-L1 cells treated with and without insulin revealed expression changes of several glycogenes. In contrast, mass spectrometric screening analysis of the glycans from these cells revealed very similar profiles suggesting that any changes in glycosylation were most likely on specific proteins rather than a global phenomenon. A fat-fed versus carbohydrate-fed mouse insulin resistant model was analysed to test the consequences of chronic insulin resistance. Muscle and liver N-glycosylation profiles from these mice are reported.
Publisher: Springer Science and Business Media LLC
Date: 29-06-2020
DOI: 10.1007/S00281-020-00801-4
Abstract: Growing evidence suggests that galectins, an evolutionarily conserved family of glycan-binding proteins, fulfill key roles in pregnancy including blastocyst implantation, maternal-fetal immune tolerance, placental development, and maternal vascular expansion, thereby establishing a healthy environment for the growing fetus. In this review, we comprehensively present the function of galectins in shaping cellular circuits that characterize a healthy pregnancy. We describe the current understanding of galectins in term and preterm labor and discuss how the galectin-glycan circuits contribute to key immunological pathways sustaining maternal tolerance and preventing microbial infections. A deeper understanding of the glycoimmune pathways regulating early events in preterm birth could offer the broader translational potential for the treatment of this devastating syndrome.
Publisher: Oxford University Press (OUP)
Date: 02-03-2007
Abstract: Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of in idual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.
Publisher: Elsevier BV
Date: 11-2018
Publisher: Elsevier BV
Date: 12-2007
Publisher: Springer Japan
Date: 20-10-2014
Publisher: Public Library of Science (PLoS)
Date: 19-12-2011
Publisher: Elsevier BV
Date: 06-2007
Publisher: Elsevier BV
Date: 07-2020
Publisher: Springer Science and Business Media LLC
Date: 09-2014
DOI: 10.1038/NG.3069
Publisher: Elsevier BV
Date: 10-2009
Publisher: Elsevier BV
Date: 07-2017
Publisher: Proceedings of the National Academy of Sciences
Date: 13-06-2006
Abstract: Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N -glycosylation. α-Mannosidase II (MII) is a key enzyme converting precursor high-mannose-type N -glycans to matured complex-type structures. Previous studies showed that MII-null mice synthesize complex-type N -glycans, indicating the presence of an alternative pathway. Because α-mannosidase IIx (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N -glycan processing by generating MII/MX double-null mice. Some double-nulls died between embryonic days 15.5 and 18.5, but most survived until shortly after birth and died of respiratory failure, which represents a more severe phenotype than that seen in single-nulls for either gene. Structural analysis of N -glycans revealed that double-nulls completely lack complex-type N -glycans, demonstrating a critical role for at least one of these enzymes for effective N -glycan processing. Recombinant mouse MX and MII showed identical substrate specificities toward N -glycan substrates, suggesting that MX is an isozyme of MII. Thus, either MII or MX can biochemically compensate for the deficiency of the other in vivo , and either of two is required for late embryonic and early postnatal development.
Publisher: Microbiology Society
Date: 06-2006
Abstract: Glycosylated proteins are ubiquitous components of eukaryote cellular surfaces, where the glycan moieties are implicated in a wide range of cell–cell recognition events. Once thought to be restricted to eukaryotes, glycosylation is now being increasingly reported in prokaryotes. Many of these discoveries have grown from advances in analytical technologies and genome sequencing. This review highlights the capabilities of high-sensitivity mass spectrometry for carbohydrate structure determination of bacterial glycoproteins and the emergence of glycoproteomic strategies that have evolved from proteomics and genomics for the functional analysis of bacterial glycosylation.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Anne Dell.