Poh-Choo Pang - Academia.edu (original) (raw)
Papers by Poh-Choo Pang
Elsevier eBooks, 2010
Mass spectrometry (MS) has proven to be the preeminent tool for the rapid, high-sensitivity analy... more Mass spectrometry (MS) has proven to be the preeminent tool for the rapid, high-sensitivity analysis of the primary structure of glycans derived from diverse biological sources including cells, fluids, secretions, tissues, and organs. These analyses are anchored by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis of permethylated derivatives of glycan pools released from the samples, to produce glycomic mass fingerprints. The application of complimentary techniques, such as chemical and enzymatic digestions, GC-MS linkage analysis, and tandem mass spectrometry (MS/MS) utilizing both electrospray (ES) and MALDI-TOF/TOF, together with bioinformatic tools allows the elucidation of incrementally more detailed structural information from the sample(s) of interest. The mouse as a model organism offers many advantages in the study of human biology, health, and disease; it is a mammal, shares 99% genetic homology with humans and its genome supports targeted mutagenesis in specific genes to produce knockouts efficiently and precisely. Glycomic analyses of tissues and organs from mice genetically deficient in one or more glycosylation gene and comparison with data collected from wild-type samples enables the facile identification of changes and perturbations within the glycome. The Consortium for Functional Glycomics (CFG) has been applying such MS-based glycomic analyses to a range of murine tissues from both wild-type and glycosylation-knockout mice in order to provide a repository of structural data for the glycobiology community. In this chapter, we describe in detail the methodologies used to prepare, derivatize, purify, and analyze glycan pools from mouse organs and tissues by MS. We also present a summary of data produced from the CFG systematic structural analysis of wild-type and knockout mouse tissues, together with a detailed example of a glycomic analysis of the Mgat4a knockout mouse.
Molecular Aspects of Medicine, Oct 1, 2016
Molecular & Cellular Proteomics, Jun 1, 2016
Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophobl... more Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. STB and CTB do not trigger histocompatibility based responses likely because they do not present any human leukocyte antigens (HLA) on their surface. This lack of HLA class I molecules could make these trophoblasts more sensitive to natural killer (NK) cell-mediated responses. However, STB and CTB are highly resistant to NK cells isolated from the decidua and peripheral blood in vitro. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary bisecting type N-glycans that have previously been implicated in the suppression of NK cell responses. Galectin-1 binds to biantennary bisecting type N-glycans. Galectins mediate many immunomodulatory activities in vitro, including the suppression of NK cell responses and promotion of the proliferation and function of inducible regulatory T cells (iTreg). These specific modifications of both adaptive and innate immune responses are considered to be essential for the development of the tolerant state in the pregnant human uterus. Extravillous cytotrophoblasts (EVT) express a paternal HLA class Ia molecule (HLA-C) on their surface, suggesting that these trophoblasts could trigger a major histocompatibility-based alloimmune response. Glycomic analysis has also confirmed the enhanced expression of carbohydrate ligands for galectins on EVT, suggesting that these trophoblasts could also sequester these immunomodulatory proteins to their surface. In summary, the specific presentation of glycans on trophoblast subpopulations supports the concept that functional glycosylation could play a role in the induction of tolerance during human pregnancy.
Journal of Virology, Feb 15, 2016
Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, d... more Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during co-evolution with their hosts, viruses have developed sophisticated mechanisms to hijack to 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 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 messenger RNAs 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 non-template process regulated by the availability of key glycosyltransferases. Interestingly, Bovine herpesvirus 4 encodes one of such enzymes 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 in function of the location and/or of the moment of the infection.
PLOS ONE, Apr 20, 2015
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species includi... more Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.
Glycobiology, Oct 27, 2007
Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ru... more 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 (NeuAcα2-3[GalNAcβ1-4]Galβ1-4GlcNAc-). Immunohistochemistry with the monoclonal 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 α2-3sialylated 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.
Journal of Bacteriology, Apr 15, 2009
Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycos... more 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 Campylobacter 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.
FEBS Letters, Mar 27, 2009
This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics,... more This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.
Springer eBooks, Oct 20, 2014
This chapter discusses mass spectrometric (MS) strategies for mammalian cell and tissue glycomics... more This chapter discusses mass spectrometric (MS) strategies for mammalian cell and tissue glycomics and places the principles underlying them within a historical framework. Ultrahigh-sensitivity MALDI-TOF/TOF glycomic methodologies are based on the analysis of permethylated derivatives of pools of glycans that are released from glycoproteins or glycolipids enzymatically or chemically. Very complex mixtures from biological extracts of cells and tissues can be screened in this way, thereby revealing the types of glycans present and, importantly, providing clues to structures that are likely to be functionally important. Optimal glycomic workflows are illustrated by glycomic data from human cytolytic T lymphocytes. Fragmentation pathways that are central to glycomics are briefly outlined, and the use of the GlycoWorkBench tool to assist fragment ion annotation is briefly explained. Finally, the open-access data S.M. Haslam (*) • P.-C. Pang • A. Antonopoulos • A. Dell Department of Life Sciences, Imperial College London, London, UK e-mail: s.haslam@imperial.ac.uk; p.pang05@imperial.ac.uk; a.antonopoulos@imperial.ac.uk; a.dell@imperial.ac.uk # Springer Japan 2015 N. Taniguchi et al. (eds.), Glycoscience: Biology and Medicine, DOI 10.1007/978-4-431-54841-6_87 69 repositories of the Consortium for Functional Glycomics, which contain glycomics data for murine and human hematopoietic cell populations, are described.
Laboratory Investigation, Jul 1, 2020
Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidual... more 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 highmannose, 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 in normal endometrium. Similar methods can be used for studies of other glycoproteins.
Diabetes, Feb 21, 2011
OBJECTIVE-Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patien... more 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.
Journal of Biological Chemistry, May 1, 2009
Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glyc... more Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetraantennary complex-type glycans carrying Gal1-4GlcNAc (lacNAc) and/or GalNAc1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAc␣2-3(GalNAc1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC. Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA), 4 glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2-5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complextype structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Gal1-4GlcNAc (lacNAc), GalNAc1-4GlcNAc (lacdiNAc), NeuAc␣2-6Gal1-4GlcNAc (sialylated lacNAc), NeuAc␣2-6GalNAc1-4GlcNAc (sialylated lacdiNAc), Gal1-4(Fuc␣1-3)GlcNAc (Lewis-x), and GalNAc1-4(Fuc␣1-3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3-5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5). Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10).
Journal of Biological Chemistry, Dec 1, 2014
Background: In vivo pharmacological inhibition of sialyltransferases has, to date, not been possi... more Background: In vivo pharmacological inhibition of sialyltransferases has, to date, not been possible. Results: 3F-NeuAc acts as a global sialyltransferase inhibitor in mice and causes kidney and liver dysfunction. Conclusion: Sialoside expression can be modulated in vivo with a sialyltransferase inhibitor. Significance: Pharmacological blockade of sialoside expression will be an important tool for future exploration of sialic acid in health and disease. Sialic acid terminates glycans of glycoproteins and glycolipids that play numerous biological roles in health and disease. Although genetic tools are available for interrogating the effects of decreased or abolished sialoside expression in mice, pharmacological inhibition of the sialyltransferase family has, to date, not been possible. We have recently shown that a sialic acid analog, 2,4,7,8,9-pentaacetyl-3F ax-Neu5Ac-CO 2 Me (3F-NeuAc), added to the media of cultured cells shuts down sialylation by a mechanism involving its intracellular conversion to CMP-3F-NeuAc, a competitive inhibitor of all sialyltransferases. Here we show that administering 3F-NeuAc to mice dramatically decreases sialylated glycans in cells of all tissues tested, including blood, spleen, liver, brain, lung, heart, kidney, and testes. A single dose results in greatly decreased sialoside expression for over 7 weeks in some tissues. Although blockade of sialylation with 3F-NeuAc does not affect viability of cultured cells, its use in vivo has a deleterious "on target" effect on liver and kidney function. After administration of 3F-NeuAc, liver enzymes in the blood are dramatically altered, and mice develop proteinuria concomitant with dramatic loss of sialic acid in the glomeruli within 4 days, leading to irreversible kidney dysfunction and failure to thrive. These results confirm a critical role for sialosides in liver and kidney function and document the feasibility of pharmacological inhibition of sialyltransferases for in vivo modulation of sialoside expression.
Biomolecules, Oct 16, 2015
Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosy... more Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed "limb-girdle CMS with tubular aggregates". CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS
Springer eBooks, 2008
There is an increasing body of evidence indicating that glycans are implicated in numerous biolog... more There is an increasing body of evidence indicating that glycans are implicated in numerous biological processes such as cell--cell interactions, intracellular signaling, and immune response. Glycomics emerges from the necessity to understand the mechanisms underlying the interactions ...
The Cell Surface, Jun 1, 2018
Link to publication on Research at Birmingham portal General rights Unless a licence is specified... more Link to publication on Research at Birmingham portal General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. • Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of 'fair dealing' under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive.
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species in-clud... more Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species in-cluding the opportunistic pathogenMycobacterium kansasii. The genome ofM. kansasii ATCC12478 contains a cluster with genes orthologous toMycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis inM. kansasii, we choseMKAN27435, a gene encoding a putative gly-cosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool pre-viously used to generate null mutants in other mycobacteria, we generated aMKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abun-dance species were detected in the mutant strain and mass spectroscopic analysis indicat-ed that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.
www.mdpi.com/journal/biomolecules/
Elsevier eBooks, 2010
Mass spectrometry (MS) has proven to be the preeminent tool for the rapid, high-sensitivity analy... more Mass spectrometry (MS) has proven to be the preeminent tool for the rapid, high-sensitivity analysis of the primary structure of glycans derived from diverse biological sources including cells, fluids, secretions, tissues, and organs. These analyses are anchored by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis of permethylated derivatives of glycan pools released from the samples, to produce glycomic mass fingerprints. The application of complimentary techniques, such as chemical and enzymatic digestions, GC-MS linkage analysis, and tandem mass spectrometry (MS/MS) utilizing both electrospray (ES) and MALDI-TOF/TOF, together with bioinformatic tools allows the elucidation of incrementally more detailed structural information from the sample(s) of interest. The mouse as a model organism offers many advantages in the study of human biology, health, and disease; it is a mammal, shares 99% genetic homology with humans and its genome supports targeted mutagenesis in specific genes to produce knockouts efficiently and precisely. Glycomic analyses of tissues and organs from mice genetically deficient in one or more glycosylation gene and comparison with data collected from wild-type samples enables the facile identification of changes and perturbations within the glycome. The Consortium for Functional Glycomics (CFG) has been applying such MS-based glycomic analyses to a range of murine tissues from both wild-type and glycosylation-knockout mice in order to provide a repository of structural data for the glycobiology community. In this chapter, we describe in detail the methodologies used to prepare, derivatize, purify, and analyze glycan pools from mouse organs and tissues by MS. We also present a summary of data produced from the CFG systematic structural analysis of wild-type and knockout mouse tissues, together with a detailed example of a glycomic analysis of the Mgat4a knockout mouse.
Molecular Aspects of Medicine, Oct 1, 2016
Molecular & Cellular Proteomics, Jun 1, 2016
Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophobl... more Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. STB and CTB do not trigger histocompatibility based responses likely because they do not present any human leukocyte antigens (HLA) on their surface. This lack of HLA class I molecules could make these trophoblasts more sensitive to natural killer (NK) cell-mediated responses. However, STB and CTB are highly resistant to NK cells isolated from the decidua and peripheral blood in vitro. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary bisecting type N-glycans that have previously been implicated in the suppression of NK cell responses. Galectin-1 binds to biantennary bisecting type N-glycans. Galectins mediate many immunomodulatory activities in vitro, including the suppression of NK cell responses and promotion of the proliferation and function of inducible regulatory T cells (iTreg). These specific modifications of both adaptive and innate immune responses are considered to be essential for the development of the tolerant state in the pregnant human uterus. Extravillous cytotrophoblasts (EVT) express a paternal HLA class Ia molecule (HLA-C) on their surface, suggesting that these trophoblasts could trigger a major histocompatibility-based alloimmune response. Glycomic analysis has also confirmed the enhanced expression of carbohydrate ligands for galectins on EVT, suggesting that these trophoblasts could also sequester these immunomodulatory proteins to their surface. In summary, the specific presentation of glycans on trophoblast subpopulations supports the concept that functional glycosylation could play a role in the induction of tolerance during human pregnancy.
Journal of Virology, Feb 15, 2016
Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, d... more Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during co-evolution with their hosts, viruses have developed sophisticated mechanisms to hijack to 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 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 messenger RNAs 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 non-template process regulated by the availability of key glycosyltransferases. Interestingly, Bovine herpesvirus 4 encodes one of such enzymes 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 in function of the location and/or of the moment of the infection.
PLOS ONE, Apr 20, 2015
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species includi... more Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.
Glycobiology, Oct 27, 2007
Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ru... more 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 (NeuAcα2-3[GalNAcβ1-4]Galβ1-4GlcNAc-). Immunohistochemistry with the monoclonal 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 α2-3sialylated 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.
Journal of Bacteriology, Apr 15, 2009
Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycos... more 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 Campylobacter 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.
FEBS Letters, Mar 27, 2009
This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics,... more This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.
Springer eBooks, Oct 20, 2014
This chapter discusses mass spectrometric (MS) strategies for mammalian cell and tissue glycomics... more This chapter discusses mass spectrometric (MS) strategies for mammalian cell and tissue glycomics and places the principles underlying them within a historical framework. Ultrahigh-sensitivity MALDI-TOF/TOF glycomic methodologies are based on the analysis of permethylated derivatives of pools of glycans that are released from glycoproteins or glycolipids enzymatically or chemically. Very complex mixtures from biological extracts of cells and tissues can be screened in this way, thereby revealing the types of glycans present and, importantly, providing clues to structures that are likely to be functionally important. Optimal glycomic workflows are illustrated by glycomic data from human cytolytic T lymphocytes. Fragmentation pathways that are central to glycomics are briefly outlined, and the use of the GlycoWorkBench tool to assist fragment ion annotation is briefly explained. Finally, the open-access data S.M. Haslam (*) • P.-C. Pang • A. Antonopoulos • A. Dell Department of Life Sciences, Imperial College London, London, UK e-mail: s.haslam@imperial.ac.uk; p.pang05@imperial.ac.uk; a.antonopoulos@imperial.ac.uk; a.dell@imperial.ac.uk # Springer Japan 2015 N. Taniguchi et al. (eds.), Glycoscience: Biology and Medicine, DOI 10.1007/978-4-431-54841-6_87 69 repositories of the Consortium for Functional Glycomics, which contain glycomics data for murine and human hematopoietic cell populations, are described.
Laboratory Investigation, Jul 1, 2020
Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidual... more 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 highmannose, 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 in normal endometrium. Similar methods can be used for studies of other glycoproteins.
Diabetes, Feb 21, 2011
OBJECTIVE-Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patien... more 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.
Journal of Biological Chemistry, May 1, 2009
Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glyc... more Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetraantennary complex-type glycans carrying Gal1-4GlcNAc (lacNAc) and/or GalNAc1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAc␣2-3(GalNAc1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC. Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA), 4 glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2-5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complextype structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Gal1-4GlcNAc (lacNAc), GalNAc1-4GlcNAc (lacdiNAc), NeuAc␣2-6Gal1-4GlcNAc (sialylated lacNAc), NeuAc␣2-6GalNAc1-4GlcNAc (sialylated lacdiNAc), Gal1-4(Fuc␣1-3)GlcNAc (Lewis-x), and GalNAc1-4(Fuc␣1-3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3-5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5). Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10).
Journal of Biological Chemistry, Dec 1, 2014
Background: In vivo pharmacological inhibition of sialyltransferases has, to date, not been possi... more Background: In vivo pharmacological inhibition of sialyltransferases has, to date, not been possible. Results: 3F-NeuAc acts as a global sialyltransferase inhibitor in mice and causes kidney and liver dysfunction. Conclusion: Sialoside expression can be modulated in vivo with a sialyltransferase inhibitor. Significance: Pharmacological blockade of sialoside expression will be an important tool for future exploration of sialic acid in health and disease. Sialic acid terminates glycans of glycoproteins and glycolipids that play numerous biological roles in health and disease. Although genetic tools are available for interrogating the effects of decreased or abolished sialoside expression in mice, pharmacological inhibition of the sialyltransferase family has, to date, not been possible. We have recently shown that a sialic acid analog, 2,4,7,8,9-pentaacetyl-3F ax-Neu5Ac-CO 2 Me (3F-NeuAc), added to the media of cultured cells shuts down sialylation by a mechanism involving its intracellular conversion to CMP-3F-NeuAc, a competitive inhibitor of all sialyltransferases. Here we show that administering 3F-NeuAc to mice dramatically decreases sialylated glycans in cells of all tissues tested, including blood, spleen, liver, brain, lung, heart, kidney, and testes. A single dose results in greatly decreased sialoside expression for over 7 weeks in some tissues. Although blockade of sialylation with 3F-NeuAc does not affect viability of cultured cells, its use in vivo has a deleterious "on target" effect on liver and kidney function. After administration of 3F-NeuAc, liver enzymes in the blood are dramatically altered, and mice develop proteinuria concomitant with dramatic loss of sialic acid in the glomeruli within 4 days, leading to irreversible kidney dysfunction and failure to thrive. These results confirm a critical role for sialosides in liver and kidney function and document the feasibility of pharmacological inhibition of sialyltransferases for in vivo modulation of sialoside expression.
Biomolecules, Oct 16, 2015
Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosy... more Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed "limb-girdle CMS with tubular aggregates". CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS
Springer eBooks, 2008
There is an increasing body of evidence indicating that glycans are implicated in numerous biolog... more There is an increasing body of evidence indicating that glycans are implicated in numerous biological processes such as cell--cell interactions, intracellular signaling, and immune response. Glycomics emerges from the necessity to understand the mechanisms underlying the interactions ...
The Cell Surface, Jun 1, 2018
Link to publication on Research at Birmingham portal General rights Unless a licence is specified... more Link to publication on Research at Birmingham portal General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. • Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of 'fair dealing' under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive.
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species in-clud... more Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species in-cluding the opportunistic pathogenMycobacterium kansasii. The genome ofM. kansasii ATCC12478 contains a cluster with genes orthologous toMycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis inM. kansasii, we choseMKAN27435, a gene encoding a putative gly-cosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool pre-viously used to generate null mutants in other mycobacteria, we generated aMKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abun-dance species were detected in the mutant strain and mass spectroscopic analysis indicat-ed that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.