Publications
249 results found
North SJ, Botchway K, Doonan J, et al., 2019, Site-specific glycoproteomic characterisation of ES-62, the major secreted product of the parasitic worm Acanthocheilonema viteae, Glycobiology, 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.
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
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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
Sweeney JG, Liang J, Antonopoulos A, et al., 2018, Loss of GCNT2/I-branched glycans enhances melanoma growth and survival, Nature Communications, Vol: 9, ISSN: 2041-1723
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.
Giovannone N, Liang J, Antonopoulos A, et al., 2018, Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans, Nature Communications, Vol: 9, ISSN: 2041-1723
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.
Shubhakar A, Pang P-C, Fernandes DL, et al., 2018, Towards automation of glycomic profiling of complex biological materials, GLYCOCONJUGATE JOURNAL, Vol: 35, Pages: 311-321, ISSN: 0282-0080
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Jankute M, Alderwick LJ, Moorey AR, et al., 2018, The singular Corynebacterium glutamicum Emb arabinofuranosyltransferase polymerises the α(1 → 5) arabinan backbone in the early stages of cell wall arabinan biosynthesis., Cell Surf, Vol: 2, Pages: 38-53
The arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae, which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (ArafTs), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ΔaftAΔemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumΔaftA, C. glutamicumΔemb, C. glutamicumΔaftAΔemb and C. glutamicumΔaftBΔaftD and two synthetic acceptors, which mimick the arabinofuranose (Araf) "primed" galactan chain, demonstrated that Cg-Emb is able to transfer an Araf residue to the C5 of the Araf positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1 → 5) ArafT and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum.
Shepherd J, Patel B, Cao H, et al., 2018, Exteriorisation of Mannoses on Human Erythrocyte Membrane Skeleton Provides 'Eat Me' Signals for Oxidatively Damaged Cells to be Cleared by Macrophages: a Pathway Mediating Hemolysis in Sickle Cell Disease, 58th Annual Scientific Meeting of the British-Society-for-Haematology, Publisher: WILEY, Pages: 119-119, ISSN: 0007-1048
Zhu F, Zhang H, Yang T, et al., 2018, Engineering and dissecting the glycosylation pathway of a streptococcal serine-rich repeat adhesion (vol 291, pg 27354, 2016), JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 293, Pages: 4952-4952, ISSN: 0021-9258
Vickers MA, Barker RN, Cao H, et al., 2017, Exteriorisation of Mannoses on Human Erythrocyte Membrane Skeleton Provides 'Eat Me' Signals for Oxidatively Damaged Cells to be Cleared By Macrophages: A Pathway Mediating Hemolysis in Sickle Cell Disease, 59th Annual Meeting of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Haslam SM, Shoulders MD, 2017, Stress, Glycomics, and Disease-A Combined Perspective, Annual Meeting of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1172-1172, ISSN: 0959-6658
Wendel U, Persson N, Risinger C, et al., 2017, Site-specific detection of advanced glycation endproducts with newly developed single-chain variable fragment antibodies, Annual Meeting of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1245-1246, ISSN: 0959-6658
Pham ND, Chang P-C, Krishnamurthy S, et al., 2017, Effects of sialic acid biosynthesis on N-linked glycan structure, cell surface interactions, and muscle diseases of aging, Annual Meeting of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1192-1192, ISSN: 0959-6658
Neelamegham S, Wang S-S, Fernandez VDS, et al., 2017, Inhibition of O-glycan biosynthesis using hexosamine analogs, Annual Meeting of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1183-1184, ISSN: 0959-6658
Termini JM, Church ES, Silver ZA, et al., 2017, Human Immunodeficiency Virus and Simian Immunodeficiency Virus Maintain High Levels of Infectivity in the Complete Absence of Mucin-Type O-Glycosylation, JOURNAL OF VIROLOGY, Vol: 91, ISSN: 0022-538X
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.
Termini JM, Silver ZA, Connor B, et al., 2017, HEK293T cell lines defective for O-linked glycosylation, PLOS One, Vol: 12, ISSN: 1932-6203
Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc) to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.
Li H, Yang T, Liao T, et al., 2017, Insights from the redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains., Microbial Cell, Vol: 4, Pages: 175-178, ISSN: 2311-2638
H. pylori is a Gram-negative extracellular bacterium, first discovered by the Australian physicians Barry Marshall and Robin Warren in 1982, that colonises the human stomach mucosa. It is the leading cause of peptic ulcer and commonly infects humans worldwide with prevalence as high as 90% in some countries. H. pylori infection usually results in asymptomatic chronic gastritis, however 10-15% of cases develop duodenal or gastric ulcers and 1-3% develop stomach cancer. Infection is generally acquired during childhood and persists for life in the absence of antibiotic treatment. H. pylori has had a long period of co-evolution with humans, going back to human migration out of Africa. This prolonged relationship is likely to have shaped the overall host-pathogen interactions and repertoire of virulence strategies which H. pylori employs to establish robust colonisation, escape immune responses and persist in the gastric niche. In this regard, H. pylori lipopolysaccharide (LPS) is a key surface determinant in establishing colonisation and persistence via host mimicry and resistance to cationic antimicrobial peptides. Thus, elucidation of the H. pylori LPS structure and corresponding biosynthetic pathway represents an important step towards better understanding of H. pylori pathogenesis and the development of novel therapeutic interventions.
Pham ND, Pang P-C, Krishnamurthy S, et al., 2017, Effects of altered sialic acid biosynthesis on N-linked glycan branching and cell surface interactions, Journal of Biological Chemistry, Vol: 292, Pages: 9637-9651, ISSN: 0021-9258
GNE myopathy is a rare muscle disorder associated with aging and is related to sporadic inclusion body myositis (sIBM), the most common acquired muscle disease of aging. While the cause of sIBM is unknown, GNE myopathy is associated with mutations in UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). GNE harbors two enzymatic activities required for biosynthesis of sialic acid in mammalian cells. Mutations to both GNE domains are linked to GNE myopathy. However, correlation between mutation-associated reductions in sialic acid production and disease severity is imperfect. To investigate other potential effects of GNE mutations, we compared sialic acid production in cell lines expressing wild-type or mutant forms of GNE. Although we did not detect any differences attributable to disease-associated mutations, lectin binding and mass spectrometry analysis revealed that GNE deficiency is associated with unanticipated effects on the structure of cell-surface glycans. In addition to exhibiting low levels of sialylation, GNE-deficient cells produced distinct N-linked glycan structures with increased branching and extended poly-N-acetyllactosamine (polyLacNAc). GNE deficiency may affect levels of UDP-GlcNAc, a key metabolite in the nutrient-sensing hexosamine biosynthetic pathway, but this modest effect did not fully account for the change in N-linked glycan structure. Further, GNE deficiency and glucose supplementation acted independently and additively to increase N-linked glycan branching. Notably, N-linked glycans produced by GNE-deficient cells displayed enhanced binding to galectin-1, indicating that changes in GNE activity can alter affinity of cell-surface glycoproteins for the galectin lattice. These findings suggest an unanticipated mechanism by which GNE activity might affect signaling through cell-surface receptors.
Li H, Yang T, Liao T, et al., 2017, The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains, PLOS Pathogens, Vol: 13, ISSN: 1553-7366
Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, s
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