Publications
302 results found
Antonopoulos A, Geddes-Sweeney JE, Dimitroff CJ, et al., 2016, Structural characterization of the N-glycome from malignant melanoma cells reveals galectin ligands, 2016 Annual Meeting of the Society for Glycobiology, Publisher: Oxford University Press, Pages: 1442-1443, ISSN: 1460-2423
Struwe WB, Agravat S, Aoki-Kinoshita KF, et al., 2016, The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets., Glycobiology, Vol: 26, Pages: 907-910, ISSN: 0959-6658
The minimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.
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.
Zhang H, Zhou M, Yang T, et al., 2016, A New Helical Binding Domain Mediates a Glycosyltransferase Activity of a Bifunctional Protein, Journal of Biological Chemistry, Vol: 291, Pages: 22106-22117, ISSN: 1083-351X
Serine-rich repeat glycoproteins (SRRPs) conserved in streptococci and staphylococci are important for bacterial colonization and pathogenesis. Fap1, a well studied SRRP is a major surface constituent of Streptococcus parasanguinis and is required for bacterial adhesion and biofilm formation. Biogenesis of Fap1 is a multistep process that involves both glycosylation and secretion. A series of glycosyltransferases catalyze sequential glycosylation of Fap1. We have identified a unique hybrid protein dGT1 (dual glycosyltransferase 1) that contains two distinct domains. N-terminal DUF1792 is a novel GT-D-type glycosyltransferase, transferring Glc residues to Glc-GlcNAc-modified Fap1. C-terminal dGT1 (CgT) is predicted to possess a typical GT-A-type glycosyltransferase, however, the activity remains unknown. In this study, we determine that CgT is a distinct glycosyltransferase, transferring GlcNAc residues to Glc-Glc-GlcNAc-modified Fap1. A 2.4-Å x-ray crystal structure reveals that CgT has a unique binding domain consisting of three α helices in addition to a typical GT-A-type glycosyltransferase domain. The helical domain is crucial for the oligomerization of CgT. Structural and biochemical studies revealed that the helix domain is required for the protein-protein interaction and crucial for the glycosyltransferase activity of CgT in vitro and in vivo. As the helix domain presents a novel structural fold, we conclude that CgT represents a new member of GT-A-type glycosyltransferases.
Mkhikian H, Mortales C-L, Zhou RW, et al., 2016, Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis, eLife, Vol: 5, Pages: 1-27, ISSN: 2050-084X
Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme β1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis.
Sweeney JG, Liang J, Giovannone N, et al., 2016, "I"-branched Carbohydrates Negatively Regulate Galectin-3-binding and Melanoma Malignancy, Journal of Immunology, Vol: 196, ISSN: 1550-6606
Giovannone N, Sweeney JG, Liang J, et al., 2016, The alpha 2,3 sialyltransferase ST3Gall regulates galectin-3 binding to human B cells, Journal of Immunology, Vol: 196, ISSN: 1550-6606
Veríssimo CM, Morassutti AL, von Itzstein M, et al., 2016, Characterization of the N-glycans of female Angiostrongylus cantonensis worms, Experimental Parasitology, Vol: 166, Pages: 137-143, ISSN: 1090-2449
Kudelka MR, Antonopoulos A, Wang Y, et al., 2016, Cellular O-Glycome Reporter/Amplification (CORA) to Explore O-Glycans of Living Cells, Experimental Biology Meeting, Publisher: FEDERATION AMER SOC EXP BIOL, ISSN: 0892-6638
Kudelka MR, Antonopoulos A, Wang Y, et al., 2016, Cellular O-Glycome Reporter/Amplification (CORA) to Explore O-Glycans of Living Cells, Experimental Biology Meeting, Publisher: FEDERATION AMER SOC EXP BIOL, ISSN: 0892-6638
Lété C, Markine-Goriaynoff N, Machiels B, et al., 2016, Bovine Herpesvirus 4 Modulates Its β-1,6-N-Acetylglucosaminyltransferase Activity through Alternative Splicing, Journal of Virology, Vol: 90, Pages: 2039-2051
Carbohydrates play major roles in host-virus interactions. It is therefore not surprising that, during coevolution with their hosts, viruses have developed sophisticated mechanisms to hijack for their profit different pathways of glycan synthesis. Thus, the Bo17 gene of Bovine herpesvirus 4 (BoHV-4) encodes a homologue of the cellular core 2 protein β-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M), which is a key player for the synthesis of complex O-glycans. Surprisingly, we show in this study that, as opposed to what is observed for the cellular enzyme, two different mRNAs are encoded by the Bo17 gene of all available BoHV-4 strains. While the first one corresponds to the entire coding sequence of the Bo17 gene, the second results from the splicing of a 138-bp intron encoding critical residues of the enzyme. Antibodies generated against the Bo17 C terminus showed that the two forms of Bo17 are expressed in BoHV-4 infected cells, but enzymatic assays revealed that the spliced form is not active. In order to reveal the function of these two forms, we then generated recombinant strains expressing only the long or the short form of Bo17. Although we did not highlight replication differences between these strains, glycomic analyses and lectin neutralization assays confirmed that the splicing of the Bo17 gene gives the potential to BoHV-4 to fine-tune the global level of core 2 branching activity in the infected cell. Altogether, these results suggest the existence of new mechanisms to regulate the activity of glycosyltransferases from the Golgi apparatus.IMPORTANCE Viruses are masters of adaptation that hijack cellular pathways to allow their growth. Glycans play a central role in many biological processes, and several studies have highlighted mechanisms by which viruses can affect glycosylation. Glycan synthesis is a nontemplate process regulated by the availability of key glycosyltransferases. Interestingly, bovine herpesvirus 4 encodes one such enzyme
Mondal N, Stolfa G, Antonopoulos A, et al., 2016, Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade, Arteriosclerosis Thrombosis and Vascular Biology, Vol: 36, Pages: 718-727, ISSN: 1524-4636
Objective—Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion.Approach and Results—A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished.Conclusions—Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.
Kudelka MR, Antonopoulos A, Wang Y, et al., 2016, Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells, Nature Methods, Vol: 13, Pages: 81-86, ISSN: 1548-7105
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.
Chen Q, Pang P-C, Cohen ME, et al., 2016, Evidence for Differential Glycosylation of Trophoblast Cell Types, Molecular & Cellular Proteomics, Vol: 15, Pages: 1857-1866
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. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3–4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2–3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2–3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered.
Pang P-C, Haslam SM, Dell A, et al., 2016, The human fetoembryonic defense system hypothesis: Twenty years on, Molecular Aspects of Medicine, Vol: 51, Pages: 71-88, ISSN: 0098-2997
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.
Aoki NP, Aoki-Kinoshita K, Agravat S, et al., 2015, The official release of the International Glycan Structure Repository, Annual Meeting of the Society-for-Glycobiology on Glycobiology - Accelerating Impact across the Biomedical Sciences, Publisher: Oxford University Press (OUP), Pages: 1279-1280, ISSN: 1460-2423
Giovannone N, Geddes-Sweeney JE, Liang J, et al., 2015, Human germinal center B cells express a unique glycosylation signature characterized by poly-N-acetyllactosaminyl glycans, Annual Meeting of the Society-for-Glycobiology on Glycobiology - Accelerating Impact across the Biomedical Sciences, Publisher: Oxford University Press (OUP), Pages: 1250-1251, ISSN: 1460-2423
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
Dewal MB, DiChiara AS, Antonopoulos A, et al., 2015, XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans, CHEMISTRY & BIOLOGY, Vol: 22, Pages: 1301-1312, ISSN: 1074-5521
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- Citations: 29
Aoki-Kinoshita K, Agravat S, Aoki NP, et al., 2015, GlyTouCan 1.0 – the international glycan structure repository, Nucleic Acids Research, Vol: 44, Pages: D1237-D1242, ISSN: 1362-4962
Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the ‘face’ of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. Therefore, the storage of glycan information in databases, let alone their curation, has been a difficult problem. This has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. As such, an international team of developers and glycobiologists have collaborated to develop this repository, called GlyTouCan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. This will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past.
Nita-Lazar M, Mancini J, Feng C, et al., 2015, The zebrafish galectins Drgal1-L2 and Drgal3-L1 bind in vitro to the infectious hematopoietic necrosis virus (IHNV) glycoprotein and reduce viral adhesion to fish epithelial cells, Developmental and Comparative Immunology, Vol: 55, Pages: 241-252, ISSN: 1879-0089
The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells. Our results suggest that the extracellular Drgal1-L2 and Drgal3-L1 interact directly and in a carbohydrate-dependent manner with the IHNV glycosylated envelope and glycans on the epithelial cell surface, significantly reducing viral adhesion.
Murray AN, Chen W, Antonopoulos A, et al., 2015, Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity, CHEMISTRY & BIOLOGY, Vol: 22, Pages: 1052-1062, ISSN: 1074-5521
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- Citations: 33
Shang C, Chen Q, Dell A, et al., 2015, The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity., PLOS One, Vol: 10, ISSN: 1932-6203
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.
Czajkowsky DM, Andersen JT, Fuchs A, et al., 2015, Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications, SCIENTIFIC REPORTS, Vol: 5, ISSN: 2045-2322
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- Citations: 28
Ersek A, Xu K, Antonopoulos A, et al., 2015, Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease, Journal of Clinical Investigation, Vol: 125, Pages: 2279-2292, ISSN: 1558-8238
Glycosphingolipids (GSLs) are essential constituents of cell membranes and lipid rafts and can modulate signal transduction events. The contribution of GSLs in osteoclast (OC) activation and osteolytic bone diseases in malignancies such as the plasma cell dyscrasia multiple myeloma (MM) is not known. Here, we tested the hypothesis that pathological activation of OCs in MM requires de novo GSL synthesis and is further enhanced by myeloma cell–derived GSLs. Glucosylceramide synthase (GCS) inhibitors, including the clinically approved agent N-butyl-deoxynojirimycin (NB-DNJ), prevented OC development and activation by disrupting RANKL-induced localization of TRAF6 and c-SRC into lipid rafts and preventing nuclear accumulation of transcriptional activator NFATc1. GM3 was the prevailing GSL produced by patient-derived myeloma cells and MM cell lines, and exogenous addition of GM3 synergistically enhanced the ability of the pro-osteoclastogenic factors RANKL and insulin-like growth factor 1 (IGF-1) to induce osteoclastogenesis in precursors. In WT mice, administration of GM3 increased OC numbers and activity, an effect that was reversed by treatment with NB-DNJ. In a murine MM model, treatment with NB-DNJ markedly improved osteolytic bone disease symptoms. Together, these data demonstrate that both tumor-derived and de novo synthesized GSLs influence osteoclastogenesis and suggest that NB-DNJ may reduce pathological OC activation and bone destruction associated with MM.
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.
Nataraj A, 2015, MKAN27435 Is Required for the Biosynthesis of Higher Subclasses of Lipooligosaccharides in Mycobacterium kansasii, PLoS ONE, Vol: 10, ISSN: 1932-6203
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.
Mondal N, Buffone A, Stolfa G, et al., 2015, ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes, Blood, Vol: 125, Pages: 687-696, ISSN: 0006-4971
The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galβ1,4GlcNAc) to create sialyl Lewis-X (sLeX) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLeX epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34+ hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.
Haslam SM, Pang PC, Antonopoulos A, et al., 2015, Mass spectrometric analyses of cell and tissue glycomes, Glycoscience: Biology and Medicine, Pages: 69-77, ISBN: 9784431548409
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 Glyco Work Bench tool to assist fragment ion annotation is briefly explained. Finally, the open-access data repositories of the Consortium for Functional Glycomics, which contain glycomics data for murine and human hematopoietic cell populations, are described.
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