265 results found
Loxley GM, Hooks DO, Antonopoulos A, et al., 2020, Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum,Trichosurus vulpecula, OPEN BIOLOGY, Vol: 10
Murphy N, Rooney B, Bhattacharyya T, et al., 2020, Glycosylation of trypanosoma cruzi TcI antigen reveals recognition by chagasic sera, Scientific Reports, Vol: 10, ISSN: 2045-2322
Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens.
Debets MF, Tastan OY, Wisnovsky SP, et al., 2020, Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 25293-25301, ISSN: 0027-8424
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase GALE like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, BH-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
Antonopoulos A, Broome S, Sharov V, et al., 2020, Site-specific characterisation of SARS-CoV-2 spike glycoprotein receptor binding domain, Glycobiology, ISSN: 0959-6658
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 characterise 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.
Sela I, Goss V, Becker-Cohen M, et al., 2020, The glycomic sialylation profile of GNE Myopathy muscle cells does not point to consistent hyposialylation of individual glycoconjugates, NEUROMUSCULAR DISORDERS, Vol: 30, Pages: 621-630, ISSN: 0960-8966
Hautala LC, Pang P-C, Antonopoulos A, et al., 2020, Altered glycosylation of glycodelin in endometrial carcinoma, Laboratory Investigation, Vol: 100, Pages: 1014-1025, ISSN: 0023-6837
Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not
Blois SM, Verlohren S, Wu G, et al., 2020, Role of galectin-glycan circuits in reproduction: from healthy pregnancy to preterm birth (PTB), Seminars in Immunopathology, Vol: 42, Pages: 469-486, ISSN: 1863-2297
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.
Osimanjiang W, Santos Roballo KC, Houck BD, et al., 2020, Analysis of N- and O-Linked Glycosylation: Differential Glycosylation after Rat Spinal Cord Injury, JOURNAL OF NEUROTRAUMA, Vol: 37, Pages: 1954-1962, ISSN: 0897-7151
Mendoza M, Lu D, Ballesteros A, et al., 2020, Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand., Glycobiology, ISSN: 0959-6658
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.
Blundell PA, Lu D, Dell A, et al., 2020, Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses, JOURNAL OF IMMUNOLOGY, Vol: 204, Pages: 1022-1034, ISSN: 0022-1767
Ibeto L, Antonopoulos A, Grassi P, et al., 2020, Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis, PLoS One, Vol: 15, ISSN: 1932-6203
Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different propo
Silver ZA, Antonopoulos A, Haslam SM, et al., 2020, Discovery of O-linked carbohydrate on HIV-1 envelope and its role in shielding against one category of broadly neutralizing antibodies, Cell Reports, Vol: 30, Pages: 1862-1869.e4, ISSN: 2211-1247
Approximately 50% of the mass of the Envelope (Env) glycoprotein surface subunit (gp120) of human immunodeficiency virus type 1 (HIV-1) is composed of N-linked carbohydrate. Until now, the dogma has been that HIV-1 lacks O-linked carbohydrate on Env. Here we show that a subset of patient-derived HIV-1 isolates contain O-linked carbohydrate on the variable 1 (V1) domain of Env gp120. We demonstrate the presence of this O-glycosylation both on virions and on gp120 expressed as a secreted protein. Further, we establish that these O-linked glycans can confer a more than 1,000-fold decrease in neutralization sensitivity (IC50) to V3-glycan broadly neutralizing antibodies. These findings uncover a structural modification to the HIV-1 Env and suggest a functional role in promoting viral escape from one category of broadly neutralizing antibodies.
Schedin-Weiss S, Gaunitz S, Sui P, et al., 2020, Glycan biomarkers for Alzheimer disease correlate with T-tau and P-tau in cerebrospinal fluid in subjective cognitive impairment, FEBS JOURNAL, Vol: 287, Pages: 3221-3234, ISSN: 1742-464X
Li H, Marceau M, Yang T, et al., 2019, East-Asian Helicobacter pylori Strains Synthesize Heptan-deficient Lipopolysaccharide, PLOS GENETICS, Vol: 15, ISSN: 1553-7404
Monzon Manzano E, Justo Sanz R, Fernandez Bello I, et al., 2019, CHANGES IN THE GLYCOSYLATION PATTERN OF THE PLATELET MEMBRANE MAY BE THE CHANGES IN THE GLYCOSYLATION PATTERN OF THE PLATELET MEMBRANE MAY BE INVOLVED IN THE PATOGENESIS OF THE PRIMARY IMMUNE THROMBOCYTOPENIA, 61st National Congress of the Spanish-Society-of-Hematology-and-Hemotherapy, Publisher: FERRATA STORTI FOUNDATION, Pages: 349-349, ISSN: 0390-6078
North SJ, Botchway K, Doonan J, et al., 2019, Site-specific glycoproteomic characterization of ES-62: The major secreted product of the parasitic worm Acanthocheilonema viteae, Glycobiology, Vol: 29, Pages: 562-571, ISSN: 0959-6658
ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of post-translational decoration by phosphorylcholine. Previously we showed that ES-62's phosphorylcholine was attached to N-linked glycans and using fast atom bombardment mass spectrometry, we characterised the structure of the glycans. However, it was unknown at this time which of ES-62's four potential N-glycosylation sites carries the phosphorylcholine-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 phosphorylcholine-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed up to two phosphorylcholine groups were detected per glycan and we are now able to characterise N-glycans with up to five phosphorylcholine groups. The number per glycan varies in three of the four glycosylation sites and in addition, for the first time, we have detected phosphorylcholine on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of phosphorylcholine 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.
Blundell PA, Lu D, Dell A, et al., 2019, Choice of host cell line is essential for the functional glycosylation of the fragment crystallizable (Fc) region of human IgG1 inhibitors of influenza B viruses
<jats:title>Abstract</jats:title><jats:p>Antibodies are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc, whose presence and fine structure profoundly impacts on their <jats:italic>in vivo</jats:italic> immunogenicity, pharmacokinetics and functional attributes. The host cell line used to produce IgG has a major impact on this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. Here we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the HEK 293-F or CHO-K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary significantly, and in particular with respect to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in CHO-K1 but not in HEK 293-F cells.</jats:p>
Lomax-Browne HJ, Robertson C, Antonopoulos A, et al., 2019, Serum IgA1 shows increased levels of α2,6-linked sialic acid in breast cancer., Interface Focus, Vol: 9, Pages: 20180079-20180079, ISSN: 2042-8898
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 samples 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 individuals. An increase in Sambucus nigra binding was observed, suggestive of increased α2,6-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 (χ2 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 samples 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.
Blundell PA, Lu D, Wilkinson M, et al., 2019, Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus, JOURNAL OF IMMUNOLOGY, Vol: 202, Pages: 1595-1611, ISSN: 0022-1767
Long JS, Mistry B, Haslam SM, et al., 2019, Host and viral determinants of influenza A virus species specificity (vol 17, pg 67, 2018), NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 124-124, ISSN: 1740-1526
Wang S-S, Gao X, Solar VD, et al., 2018, Thioglycosides Are efficient metabolic decoys of glycosylation that reduce selectin dependent leukocyte adhesion, Cell Chemical Biology, Vol: 25, Pages: 1-14, ISSN: 2451-9448
Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10-100 μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyte rolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ∼80%-90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation.
Giovannone N, Antonopoulos A, Liang J, et al., 2018, Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans, FRONTIERS IN IMMUNOLOGY, Vol: 9, ISSN: 1664-3224
El Jellas K, Johansson BB, Fjeld K, et al., 2018, The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 293, Pages: 19476-19491, ISSN: 0021-9258
Dell A, Lu D, Haslam SM, et al., 2018, Towards a novel cancer vaccine: Characterisation of the glycome of canine melanoma cells, Annual Meeting of the Society-for-Glycobiology (SFG), Publisher: OXFORD UNIV PRESS INC, Pages: 1038-1039, ISSN: 0959-6658
Cao H, Wassall HJ, Forrester MA, et al., 2018, Hemoglobin S induces exposure of red blood cell membrane skeleton microdomains bearing mannose that stimulate phagocytosis by macrophages: A molecular basis for hemolysis in sickle cell disease but protection against Plasmodium Falciparum malaria, 60th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: American Society of Hematology, ISSN: 1528-0020
Monzon Manzano E, Justo Sanz R, Haslam SM, et al., 2018, Platelet Protein Glycosylation in Immune Thrombocytopenia, 60th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Long JS, Mistry B, Haslam SM, et al., 2018, Host and viral determinants of influenza A virus species specificity, NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 67-81, ISSN: 1740-1526
El Jellas K, Haslam SM, Choi MH, et al., 2018, Glycosylation profiles and ABO blood group antigens of the Carboxyl-ester Lipase (CEL) protein associated with chronic pancreatitis and MODY8 syndrome, 49th Annual Meeting of the American Pancreatic Association, Publisher: Lippincott, Williams & Wilkins, Pages: 1396-1396, ISSN: 0885-3177
Wong MY, Chen K, Antonopoulos A, et al., 2018, XBP1s activation can globally remodel N-glycan structure distribution patterns, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 115, Pages: E10089-E10098, ISSN: 0027-8424
Veerapen N, Kharkwal SS, Jervis P, et al., 2018, Photoactivable Glycolipid Antigens Generate Stable Conjugates with CD1d for Invariant Natural Killer T Cell Activation, BIOCONJUGATE CHEMISTRY, Vol: 29, Pages: 3161-3173, ISSN: 1043-1802
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