Publications
495 results found
Brune KD, Lieknina I, Sutov G, et al., 2021, N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone, CHEMBIOCHEM, Vol: 22, Pages: 3199-3207, ISSN: 1439-4227
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- Citations: 2
Antonopoulos A, Broome S, Sharov V, et al., 2021, Site-specific characterisation of SARS-CoV-2 spike glycoprotein receptor binding domain, Glycobiology, Vol: 31, Pages: 181-187, 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.
Ghedini M, Caprioli RM, Cooks GR, et al., 2021, Giovanni Sindona reminiscences, JOURNAL OF MASS SPECTROMETRY, Vol: 56, ISSN: 1076-5174
Hunt R, Hettiarachchi G, Katneni U, et al., 2019, A single synonymous variant (c.354G > A [p.P118P]) in ADAMTS13 confers enhanced specific activity, International Journal of Molecular Sciences, Vol: 20, Pages: 1-18, ISSN: 1422-0067
Synonymous variants within coding regions may influence protein expression and function. We have previously reported increased protein expression levels ex vivo (~120% in comparison to wild-type) from a synonymous polymorphism variant, c.354G>A [p.P118P], of the ADAMTS13 gene, encoding a plasma protease responsible for von Willebrand Factor (VWF) degradation. In the current study, we investigated the potential mechanism(s) behind the increased protein expression levels from this variant and its effect on ADAMTS13 physico-chemical properties. Cell-free assays showed enhanced translation of the c.354G>A variant and the analysis of codon usage characteristics suggested that introduction of the frequently used codon/codon pair(s) may have been potentially responsible for this effect. Limited proteolysis, however, showed no substantial influence of altered translation on protein conformation. Analysis of post-translational modifications also showed no notable differences but identified three previously unreported glycosylation markers. Despite these similarities, p.P118P variant unexpectedly showed higher specific activity. Structural analysis using modeled interactions indicated that subtle conformational changes arising from altered translation kinetics could affect interactions between an exosite of ADAMTS13 and VWF resulting in altered specific activity. This report highlights how a single synonymous nucleotide variation can impact cellular expression and specific activity in the absence of measurable impact on protein structure.
Richards E, Bouché L, Panico M, et al., 2018, The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology., Journal of Biological Chemistry, Vol: 293, Pages: 18123-18137, ISSN: 0021-9258
Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficilestrain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using mass spectrometry and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide, (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-, and (iii) a non-reducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24 kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile. In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficilesporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.
Bouché L, Panico M, Hitchen P, et al., 2016, The Type B flagellin of hypervirulent Clostridium difficile is modified with novel sulphonated Peptidylamido-glycans, Journal of Biological Chemistry, Vol: 291, Pages: 25439-25449, ISSN: 1083-351X
Glycosylation of flagellins is a well recognized property of many bacterial species. In this study we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. We used mass spectrometry (nano LC- MS and MS/MS analysis) to identify a number of putative glycopeptides which carried a variety of glycoform substitutions each of which was linked through an initial HexNAc residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of Electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive- and negative-ion collisionally activated decomposition (CAD) MS/MS which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a Taurine-containing peptidylamido-glycan structure. Finally NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-β-GlcNAc-(1→)Ser and the novel capping moiety was shown to be comprised of Taurine, Alanine, and Glycine. This is the first report of a novel O-linked sulphonated peptidylamido-glycan moiety decorating a flagellin protein.
Valiente E, Bouche L, Hitchen P, et al., 2016, Role of glycosyltransferases modifying type B flagellin of emerging hypervirulent Clostridium difficile lineages and their impact on motility and biofilm formation, Journal of Biological Chemistry, Vol: 291, Pages: 25450-25461, ISSN: 1083-351X
Clostridium difficile is the principal cause of nosocomial infectious diarrhea worldwide. The pathogen modifies its flagellin with either a type A or type B O-linked glycosylation system, which has a contributory role in pathogenesis. We study the functional role of glycosyltransferases modifying type B flagellin in the 023 and 027 hypervirulent C. difficile lineages by mutagenesis of five putative glycosyltransferases and biosynthetic genes. We reveal their roles in the biosynthesis of the flagellin glycan chain and demonstrate that flagellar post-translational modification affects motility and adhesion-related bacterial properties of these strains. We show that the glycosyltransferases 1 and 2 (GT1 and GT2) are responsible for the sequential addition of a GlcNAc and two rhamnoses, respectively, and that GT3 is associated with the incorporation of a novel sulfonated peptidyl-amido sugar moiety whose structure is reported in our accompanying paper (Bouché, L., Panico, M., Hitchen, P., Binet, D., Sastre, F., Faulds-Pain, A., Valiente, E., Vinogradov, E., Aubry, A., Fulton, K., Twine, S., Logan, S. M., Wren, B. W., Dell, A., and Morris, H. R. (2016) J. Biol. Chem. 291, 25439–25449). GT2 is also responsible for methylation of the rhamnoses. Whereas type B modification is not required for flagellar assembly, some mutations that result in truncation or abolition of the glycan reduce bacterial motility and promote autoaggregation and biofilm formation. The complete lack of flagellin modification also significantly reduces adhesion of C. difficile to Caco-2 intestinal epithelial cells but does not affect activation of human TLR5. Our study advances our understanding of the genes involved in flagellar glycosylation and their biological roles in emerging hypervirulent C. difficile strains.
Panico M, Bouché L, Binet D, et al., 2016, Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding, Scientific Reports, Vol: 6, Pages: 1-17, ISSN: 2045-2322
The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU 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.
Wu G, Hitchen PG, Panico M, et al., 2015, Glycoproteomic studies of IgE from a novel hyper IgE syndrome linked to PGM3 mutation, Glycoconjugate Journal, Vol: 33, Pages: 447-456, ISSN: 1573-4986
Glycans serve as important regulators of antibody activities and half-lives. IgE is the most heavily glycosylated antibody, but in comparison to other antibodies little is known about its glycan structure function relationships. We therefore describe the site specific IgE glycosylation from a patient with a novel hyper IgE syndrome linked to mutations in PGM3, which is an enzyme involved in synthesizing UDP-GlcNAc, a sugar donor widely required for glycosylation. A two-step method was developed to prepare two IgE samples from less than 1 mL of serum collected from a patient with PGM3 mutation and a patient with atopic dermatitis as a control subject. Then, a glycoproteomic strategy was used to study the site-specific glycosylation. No glycosylation was found at Asn264, whilst high mannose glycans were only detected at Asn275, tri-antennary glycans were exclusively observed at Asn99 and Asn252, and non-fucosylated complex glycans were detected at Asn99. The results showed similar glycosylation profiles between the two IgE samples. These observations, together with previous knowledge of IgE glycosylation, imply that IgE glycosylation is similarly regulated among healthy control, allergy and PGM3 related hyper IgE syndrome.
Dell A, Morris HR, Panico M, et al., 2015, N- and O-glycosylation of gp120 isolated from HIV virions, Glycobiology: accelerating impact across the biomedical sciences, Publisher: Oxford University Press (OUP), Pages: 1230-1230, ISSN: 1460-2423
Morris H, Reason A, 2015, Compare and contrast, EBR - European Biopharmaceutical Review, Pages: 18-24, ISSN: 1369-0663
A range of analytical techniques can be used to prove that the structural integrity of an active pharmaceutical biological ingredient has been retained following any necessary purification. These analyses should assess the intactness, protein backbone, PTMs, oligosaccharide profile and secondary/tertiary structure of the product, pre- and post-purification.
Stansell A, 2015, Gp120 on HIV-1 Virions Lacks O-Linked Carbohydrate, PLOS ONE, Vol: 10, Pages: 1-15
As HIV-1-encoded envelope protein traverses the secretory pathway, it may be modified with N- and O-linked carbohydrate. When the gp120s of HIV-1 NL4-3, HIV-1 YU2, HIV-1 Bal, HIV-1 JRFL, and HIV-1 JRCSF were expressed as secreted proteins, the threonine at consensus position 499 was found to be O-glycosylated. For SIVmac239, the corresponding threonine was also glycosylated when gp120 was recombinantly expressed. Similarly-positioned, highly-conserved threonines in the influenza A virus H1N1 HA1 and H5N1 HA1 envelope proteins were also found to carry O-glycans when expressed as secreted proteins. In all cases, the threonines were modified predominantly with disialylated core 1 glycans, together with related core 1 and core 2 structures. Secreted HIV-1 gp140 was modified to a lesser extent with mainly monosialylated core 1 O-glycans, suggesting that the ectodomain of the gp41 transmembrane component may limit the accessibility of Thr499 to glycosyltransferases. In striking contrast to these findings, gp120 on purified virions of HIV-1 Bal and SIV CP-MAC lacked any detectable O-glycosylation of the C-terminal threonine. Our results indicate the absence of O-linked carbohydrates on Thr499 as it exists on the surface of virions and suggest caution in the interpretation of analyses of post-translational modifications that utilize recombinant forms of envelope protein.
Morris HR, 2015, Howard Redfern Morris, The Encyclopedia of Mass Spectrometry: Volume 9: Historical Perspectives, Part B: Notable People in Mass Spectrometry, Pages: 159-160, ISBN: 9780081003954
Haslam S, Panico M, Morris H, et al., 2013, Glycomics and glycoproteomics-providing new biological insights, Annual Conference of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1330-1330, ISSN: 0959-6658
Meyer BH, Peyfoon E, Dietrich C, et al., 2013, Agl16, a Thermophilic Glycosyltransferase Mediating the Last Step of <i>N</i>-Glycan Biosynthesis in the Thermoacidophilic Crenarchaeon <i>Sulfolobus acidocaldarius</i>, JOURNAL OF BACTERIOLOGY, Vol: 195, Pages: 2177-2186, ISSN: 0021-9193
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- Citations: 31
Canis K, McKinnon TAJ, Nowak A, et al., 2012, Mapping the N-glycome of human von Willebrand factor, BIOCHEMICAL JOURNAL, Vol: 447, Pages: 217-228, ISSN: 0264-6021
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- Citations: 76
Harrison R, Hitchen PG, Panico M, et al., 2012, Glycoproteomic characterization of recombinant mouse α-dystroglycan, GLYCOBIOLOGY, Vol: 22, Pages: 662-675, ISSN: 0959-6658
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- Citations: 46
Clark GF, Grassi P, Pang P, et al., 2012, Tumor Biomarker Glycoproteins in the Seminal Plasma of Healthy Human Males Are Endogenous Ligands for DC-SIGN, Molecular & Cellular Proteomics, Vol: 11
DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewisx and Lewisy carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewisx and Lewisy sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.
Meyer BH, Zolghadr B, Peyfoon E, et al., 2011, Sulfoquinovose synthase - an important enzyme in the N-glycosylation pathway of Sulfolobus acidocaldarius, MOLECULAR MICROBIOLOGY, Vol: 82, Pages: 1150-1163, ISSN: 0950-382X
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- Citations: 61
Stansell E, Canis K, Huang I-C, et al., 2011, O-Glycosylation of the Envelope Glycoprotein of the Human Immunodeficiency Virus, Annual Conference of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1454-1454, ISSN: 0959-6658
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- Citations: 1
Pang P, Chiu PCN, Lee C, et al., 2011, Human Sperm Binding Is Mediated by the Sialyl-Lewisx Oligosaccharide on the Zona Pellucida, Science, Vol: 333, Pages: 1761-1764
Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewisx sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewisx sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewisx sequence represents the major carbohydrate ligand for human sperm-egg binding.
Hayee B, Antonopoulos A, Murphy EJ, et al., 2011, <i>G6PC3</i> mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction, GLYCOBIOLOGY, Vol: 21, Pages: 914-924, ISSN: 0959-6658
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- Citations: 69
Ahnstrom J, Andersson HM, Canis K, et al., 2011, Activated protein S cofactor function of protein S: a novel function for a γ-carboxyglutamic acid residue, Publisher: WILEY-BLACKWELL, Pages: 14-14, ISSN: 1538-7933
Ahnstroem J, Andersson HM, Canis K, et al., 2011, Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue, BLOOD, Vol: 117, Pages: 6685-6693, ISSN: 0006-4971
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- Citations: 31
Lee C, Chiu PCN, Pang P, et al., 2011, Glycosylation Failure Extends to Glycoproteins in Gestational Diabetes Mellitus: Evidence From Reduced α2-6 Sialylation and Impaired Immunomodulatory Activities of Pregnancy-Related Glycodelin-A, Diabetes, Vol: 60, Pages: 909-917
OBJECTIVE 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.RESEARCH DESIGN AND METHODS 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.RESULTS 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.CONCLUSIONS Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.
Clark G, Pang P-C, Grassi P, et al., 2010, Tumor Biomarker Glycoproteins Present in the Seminal Plasma of Healthy Human Males Are Novel Endogenous Ligands for DC-Sign, Annual Conference of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1522-1522, ISSN: 0959-6658
Dell A, Morris H, Haslam S, 2010, Functional Glycomics: The Pivotal Role of Mass Spectrometry, Annual Conference of the Society-for-Glycobiology, Publisher: OXFORD UNIV PRESS INC, Pages: 1450-1450, ISSN: 0959-6658
Jervis AJ, Langdon R, Hitchen P, et al., 2010, Characterization of N-Linked Protein Glycosylation in <i>Helicobacter pullorum</i>, JOURNAL OF BACTERIOLOGY, Vol: 192, Pages: 5228-5236, ISSN: 0021-9193
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- Citations: 53
Harris EN, Parry S, Sutton-Smith M, et al., 2010, <i>N-</i>Glycans on the link domain of human HARE/Stabilin-2 are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan, GLYCOBIOLOGY, Vol: 20, Pages: 991-1001, ISSN: 0959-6658
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- Citations: 14
Hitchen P, Brzostek J, Panico M, et al., 2010, Modification of the <i>Campylobacter jejuni</i> flagellin glycan by the product of the Cj1295 homopolymeric-tract-containing gene, MICROBIOLOGY-SGM, Vol: 156, Pages: 1953-1962, ISSN: 1350-0872
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- Citations: 42
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