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Silva JF, Olivon VC, Mestriner FLAC, Zanotto CZ, Ferreira RG, Ferreira NS, Silva CAA, Luiz JPM, Alves JV, Fazan R, Cunha FQ, Alves-Filho JC, Tostes RC. Acute Increase in O-GlcNAc Improves Survival in Mice With LPS-Induced Systemic Inflammatory Response Syndrome. Frontiers in physiology 2019 10 32038294
Abstract:
Sepsis is a systemic inflammatory response syndrome (SIRS) resulting from a severe infection that is characterized by immune dysregulation, cardiovascular derangements, and end-organ dysfunction. The modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) influences many of the key processes that are altered during sepsis, including the production of inflammatory mediators and vascular contractility. Here, we investigated whether O-GlcNAc affects the inflammatory response and cardiovascular dysfunction associated with sepsis. Mice received an intraperitoneal injection of lipopolysaccharide (LPS, 20 mg/Kg) to induce endotoxic shock and systemic inflammation, resembling sepsis-induced SIRS. The effects of an acute increase in O-GlcNAcylation, by treatment of mice with glucosamine (GlcN, 300 mg/Kg, i.v.) or thiamet-G (ThG, 150 μg/Kg, i.v.), on LPS-associated mortality, production and release of cytokines by macrophages and vascular cells, vascular responsiveness to constrictors and blood pressure were then determined. Mice under LPS-induced SIRS exhibited a systemic and local inflammatory response with increased levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), as well as severe hypotension and vascular hyporesponsiveness, characterized by reduced vasoconstriction to phenylephrine. In addition, LPS increased neutrophil infiltration in lungs and produced significant lethality. Treatment with GlcN and ThG reduced systemic inflammation and attenuated hypotension and the vascular refractoriness to phenylephrine, improving survival. GlcN and ThG also decreased LPS-induced production of inflammatory cytokines by bone marrow-derived macrophages and nuclear transcription factor-kappa B (NF-κB) activation in RAW 264.7 NF-κB promoter macrophages. Treatment of mice with ThG increased O-glycosylation of NF-κB p65 subunit in mesenteric arteries, which was associated with reduced Ser536 phosphorylation of NF-κB p65. Finally, GlcN also increased survival rates in mice submitted to cecal ligation and puncture (CLP), a sepsis model. In conclusion, increased O-GlcNAc reduces systemic inflammation and cardiovascular disfunction in experimental sepsis models, pointing this pathway as a potential target for therapeutic intervention.
O-GlcNAc proteins:
CRYAA, TF65
Species: Bos taurus, Mus musculus
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Tsumoto H, Akimoto Y, Endo T, Miura Y. Quantitative analysis of O-GlcNAcylation in combination with isobaric tag labeling and chemoenzymatic enrichment. Bioorganic & medicinal chemistry letters 2017 27(22) 29029932
Abstract:
Protein O-GlcNAcylation regulates various biological processes, and is associated with several diseases. Therefore, the development of quantitative proteomics is important for understanding the mechanisms of O-GlcNAc-related diseases. We previously reported selective enrichment of O-GlcNAcylated peptides, which provided high-selectivity and effective release by a novel thiol-alkyne and thiol-disulfide exchange. Here, we describe a new approach using initial isobaric tag labeling for relative quantification followed by enrichment and β-elimination/Michael addition with dithiothreitol for identification of both proteins and modification sites. The approach was validated using model proteins and peptides. This novel strategy could be used for quantitative O-GlcNAcome of biological samples.
O-GlcNAc proteins:
CRYAA
Species: Bos taurus
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Bunkenborg J, Falkenby LG, Harder LM, Molina H. Covalent perturbation as a tool for validation of identifications and PTM mapping applied to bovine alpha-crystallin. Proteomics 2016 16(4) 26644245
Abstract:
Proteomic identifications hinge on the measurement of both parent and fragment masses and matching these to amino acid sequences via database search engines. The correctness of the identifications is assessed by statistical means. Here we present an experimental approach to test identifications. Chemical modification of all peptides in a sample leads to shifts in masses depending on the chemical properties of each peptide. The identification of a native peptide sequence and its perturbed version with a different parent mass and fragment ion masses provides valuable information. Labeling all peptides using reductive alkylation with formaldehyde is one such perturbation where the ensemble of peptides shifts mass depending on the number of reactive amine groups. Matching covalently perturbed fragmentation patterns from the same underlying peptide sequence increases confidence in the assignments and can salvage low scoring post-translationally modified peptides. Applying this strategy to bovine alpha-crystallin, we identify 9 lysine acetylation sites, 4 O-GlcNAc sites and 13 phosphorylation sites.
O-GlcNAc proteins:
CRYAA, CRYAB
Species: Bos taurus
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Nishikaze T, Kawabata S, Iwamoto S, Tanaka K. Reversible hydrazide chemistry-based enrichment for O-GlcNAc-modified peptides and glycopeptides having non-reducing GlcNAc residues. The Analyst 2013 138(23) 24131013
Abstract:
O-Linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translational modification (PTM) of proteins. Analysis of O-GlcNAc modification using mass spectrometry (MS) is often problematic because of the low stoichiometry of the modification. In this study, we developed a new method for enriching O-GlcNAc-modified peptides using reversible hydrazide chemistry. O-GlcNAc-modified peptides were first labeled with N-azidoacetylgalactosamine (GalNAz) using gatactosyltransferase-T1 (Y289L) enzyme. The azide group on the GalNAz residue was then reacted with 3-ethynylbenzaldehyde via copper-catalyzed Huisgen 1,3-cycloaddition "click reaction" to form an aromatic aldehyde group of glycopeptides. Aromatic aldehyde-derivatized glycopeptides were enriched by reversible hydrazone formation with hydrazide resin. Reaction conditions for each step, especially for the click reaction, were optimized to achieve complete reaction without significant side reactions. This method was validated using a tryptic digest of bovine α-crystallin, which is an O-GlcNAc-modified glycoprotein. The developed method was also applied to structure-specific enrichment of N-linked glycopeptides having non-reducing terminal GlcNAc residues. All materials and chemicals required for this method are commercially available and there is no need to prepare any special reagents, facilitating the introduction of this method in any laboratory.
O-GlcNAc proteins:
CRYAA
Species: Bos taurus
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Overath T, Kuckelkorn U, Henklein P, Strehl B, Bonar D, Kloss A, Siele D, Kloetzel PM, Janek K. Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag. Molecular & cellular proteomics : MCP 2012 11(8) 22556278
Abstract:
The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.
O-GlcNAc proteins:
PSB1, CRYAA, HS90A, HS90B, PSA6, PSA7, PSA5
Species: Bos taurus, Mus musculus
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Klement E, Lipinszki Z, Kupihár Z, Udvardy A, Medzihradszky KF. Enrichment of O-GlcNAc modified proteins by the periodate oxidation-hydrazide resin capture approach. Journal of proteome research 2010 9(5) 20146544
Abstract:
A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion, and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation, the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.
O-GlcNAc proteins:
B7Z0D3, CRYAA, MA205, FAF, SCRIB
Species: Drosophila melanogaster, Bos taurus
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Viner RI, Zhang T, Second T, Zabrouskov V. Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem mass tags and electron transfer dissociation. Journal of proteomics 2009 72(5) 19245863
Abstract:
The modification of Ser/Thr residues in proteins by addition of single O-linked N-acetylglucosamine (O-GlcNAc) moieties play an important role in cell regulation. However, understanding the cellular mechanisms that regulate O-GlcNAc glycosylation has been challenging due to the difficulty in detection and quantification of this modification. Mass spectrometry-based multiplex quantitative approaches have been successfully employed to measure relative phosphorylation levels using collisionally induced dissociation (CID). However, labile modifications such as O-GlcNAc are lost prior to fragmentation of the peptide backbone in conventional CID, often preventing correct peptide identification, localization of the modified site, and as a result, relative quantification. Compared to CID, Electron Transfer Dissociation (ETD) preserves labile post-translational modifications (PTMs), and allows direct mapping of peptide/protein modifications. This is the first report to assess the utility of combining multiplexed isobaric tandem mass tag (TMT) labeling and ETD for relative quantification of labile PTMs. ETD analysis of both labeled and unlabeled peptides from bovine alpha-crystallins pinpointed at least one O-GlcNAc containing modification site in each of the protein subunits, in addition to a multitude of other PTMs, including glycation, phosphorylation, and acetylation. Moreover, ETD of TMT(6) labeled peptides produced four unique reporter ions that could be used for relative quantification. TMT reporter ion ratios measured by ETD had similar accuracy and precision as those obtained by conventional CID techniques. When applied to glycosylated or otherwise modified peptides, ETD was the only dissociation method which consistently provided confident sequence identification, PTM localization, and quantitative information, all in the same spectrum. This suggests that ETD-based workflows can be complementary to traditional CID approaches when used for simultaneous qualitative and quantitative analysis of modified peptides.
O-GlcNAc proteins:
CRYAA, CRYAB
Species: Bos taurus
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Dehennaut V, Slomianny MC, Page A, Vercoutter-Edouart AS, Jessus C, Michalski JC, Vilain JP, Bodart JF, Lefebvre T. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Molecular & cellular proteomics : MCP 2008 7(11) 18617508
Abstract:
O-Linked N-acetylglucosaminylation (O-GlcNAcylation) (or O-linked N-acetylglucosamine (O-GlcNAc)) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have described previously the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G(2)/M transition, and we have demonstrated that the inhibition of O-GlcNAc-transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we identified proteins that are O-GlcNAc-modified during the G(2)/M transition. Because of a low expression of O-GlcNAcylation in Xenopus oocyte, classical enrichment of O-GlcNAc-bearing proteins using O-GlcNAc-directed antibodies or wheat germ agglutinin lectin affinity were hard to apply, albeit these techniques allowed the identification of actin and erk2. Therefore, another strategy based on an in vitro enzymatic labeling of O-GlcNAc residues with azido-GalNAc followed by a chemical addition of a biotin alkyne probe and by enrichment of the tagged proteins on avidin beads was used. Bound proteins were analyzed by nano-LC-nano-ESI-MS/MS allowing for the identification of an average of 20 X. laevis oocyte O-GlcNAcylated proteins. In addition to actin and beta-tubulin, we identified metabolic/functional proteins such as PP2A, proliferating cell nuclear antigen, transitional endoplasmic reticulum ATPase, aldolase, lactate dehydrogenase, and ribosomal proteins. This labeling allowed for the mapping of a major O-GlcNAcylation site within the 318-324 region of beta-actin. Furthermore immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomally bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification both on microtubules and on chromosomes remains to be determined. However, the mapping of the O-GlcNAcylation sites will help to underline the function of this post-translational modification on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle.
O-GlcNAc proteins:
ACTB, RS31, CRYAA, RL4A, ENOA, RL18A, RL5A, EF1A2, PCNA, TERA, MK01, TBB4, LDHB, LDHA, RS32, G3P, SAHHA, RAN, Q5XGY5, Q7SZ23, STIP1, Q8AVH2, KPYM, Q92140
Species: Xenopus laevis, Bos taurus
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Clark PM, Dweck JF, Mason DE, Hart CR, Buck SB, Peters EC, Agnew BJ, Hsieh-Wilson LC. Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. Journal of the American Chemical Society 2008 130(35) 18683930
Abstract:
We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.
O-GlcNAc proteins:
A0A0G2JYI7, A0A0G2K931, A0A0G2KAS9, ROA2, OTUB1, SEP11, B5DFG5, D3ZM69, HCFC1, D3ZQM0, D3ZUY8, D3ZVQ0, D4A0B4, D4A133, D4A1L2, D4A554, D4AA63, D4ABT8, D4ACB8, E9PT29, E9PTR4, S2512, RPGF2, F1M3L4, F1M3W5, F1M7Y3, F1M8R0, F1M949, F1M9V7, G3V7M0, G3V8Q8, HNRPC, SERA, AMPH, DCLK1, NCDN, CTND2, DNM1L, PICAL, CLIP2, ANK3, PACS1, HSP74, BSN, PTN23, AATM, CRYAA, ROA1, ENOA, HXK1, ENOG, ODP2, ALDOC, GLNA, SYN1, SORL, PLCB1, ATPB, DHE3, KCC2A, AT2A2, PDIA3, KPYM, FAS, AATC, MTAP2, MAP1B, ATPA, SPTN1, PGK1, HMCS1, AP2A2, CSK21, PO3F1, DYN1, MK03, AINX, THOP1, PGAM1, VPP1, ODPA, DCTN1, MARCS, MAP1A, 1433B, DYHC1, TERA, RP3A, PFKAM, PFKAP, ANXA6, TPIS, ODPB, NU153, GDIA, HUWE1, PYGB, GBB1, SNAA, NCKP1, OGT1, ACTB, HNRPK, 1433G, RS8, 1433E, RL7A, EF1A1, ACTA, VATB2, RS3, AP2B1, CH60, RACK1, PP2BA, KAPCB, 1433T, EF2K, VDAC2, GNAQ, TBB2A, FSCN1, ARPC2, CAND1, AP180, ADT1, ANXA2, ADT2, MYPT1, MIC60, TBB2B, CARM1, SRPRB, RD23B, TBB3, RBMX, OXR1, Q4V8F6, ZFR, WASF1, E41L5, RUFY3, DTBP1, SHLB2, Q5RJQ1, WDR1, IF4A2, CAPZB, ODO1, TBA4A, TCPB, SPTB2, 4EBP1, CAPS1, SYNJ1, DPYL1, DPYL4, DPYL3, CAMKV, KCC1A, MAP6, NDUS2, BACH, HS105, NDUS1, MAVS, PURB, QCR1, Q6AY07, Q6AYI1, PLAK, TBB4B, ROA3, Q6XLI7, PGAP1, FMR1, RB6I2, COR1A, IDH3A, PABP1, TALDO, DPYL5, WNK1, RIMS1, SEPT5, UBQL1, PCLO, MYH10, Q9JMB3, NSF, FKBP4, SHAN2, SRCN1, MYO5A, CRYM, GORS2, E41L1, GUAD, SEPT7, PACN1, Q9Z1X8, HOME1, GIT1, CTBP1, SC31A
Species: Rattus norvegicus, Bos taurus, Mus musculus
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Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proceedings of the National Academy of Sciences of the United States of America 2004 101(36) 15340146
Abstract:
The covalent modification of intracellular proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as a crucial regulatory posttranslational modification akin to phosphorylation. Numerous studies point to the significance of O-GlcNAc in cellular processes such as nutrient sensing, protein degradation, and gene expression. Despite its importance, the breadth and functional roles of O-GlcNAc are only beginning to be elucidated. Advances in our understanding will require the development of new strategies for the detection and study of O-GlcNAc-modified proteins in vivo. Herein we report the direct, high-throughput analysis of O-GlcNAc-glycosylated proteins from the mammalian brain. The proteins were identified by using a chemoenzymatic approach that exploits an engineered galactosyltransferase enzyme to selectively label O-GlcNAc proteins with a ketone-biotin tag. The tag permits enrichment of low-abundance O-GlcNAc species from complex mixtures and localization of the modification to short amino acid sequences. Using this approach, we discovered 25 O-GlcNAc-glycosylated proteins from the brain, including regulatory proteins associated with gene expression, neuronal signaling, and synaptic plasticity. The functional diversity represented by this set of proteins suggests an expanded role for O-GlcNAc in regulating neuronal function. Moreover, the chemoenzymatic strategy described here should prove valuable for identifying O-GlcNAc-modified proteins in various tissues and facilitate studies of the physiological significance of O-GlcNAc across the proteome.
O-GlcNAc proteins:
A3E0T0, HCFC1, D4A3Q3, D4A543, RPGF2, BSN, CRYAA, MTAP2, MAP1B, ATF2, TLE4, Q498M7, AGFG1, ZFR, NFRKB, WNK1, SPTN2, GORS2, E41L1, SYNPO
Species: Rattus norvegicus, Bos taurus, Mus musculus
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Roquemore EP, Dell A, Morris HR, Panico M, Reason AJ, Savoy LA, Wistow GJ, Zigler JS Jr, Earles BJ, Hart GW. Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine. The Journal of biological chemistry 1992 267(1) 1730617
Abstract:
Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.
O-GlcNAc proteins:
F7H3G0, CRYAA, CRYAA, CRYAA, CRYAA, CRYAB, CRYAB
Species: Macaca mulatta, Homo sapiens, Bos taurus, Rhea americana
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