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ESSENTIALS OF GLYCOBIOLOGY LECTURE 15 The O-GlcNAc Modification Hud Freeze. ADAPTED FROM. “Dynamic Interplay Between O-GlcNAc and O-Phosphate: Roles in Transcription, Signaling and in Cellular Response to Stress.”. Gerald W. Hart Johns Hopkins University School of Medicine. O-GlcNAc.
ESSENTIALS OF GLYCOBIOLOGYLECTURE 15The O-GlcNAc ModificationHud FreezeADAPTED FROM“Dynamic Interplay Between O-GlcNAc and O-Phosphate: Roles in Transcription, Signaling and in Cellular Response to Stress.”Gerald W. HartJohns Hopkins UniversitySchool of MedicineO-GlcNAcO- Linked N-AcetylglucosamineO-GlcNAc TransferaseO-GlcNAcaseA dynamic post-translational modificationOHOHHOHOOOOOHOHOAcNHAcNHTyr-Ser-Pro-Thr-Ser-Pro-SerKey Features of O-GlcNAc :
  • NOT elongated to more complex structures.
  • Localized to the cytoplasm and nucleus.
  • Present in all higher eukaryotes studied.
  • As abundant as phosphorylation;
  • UDP-GlcNAc is Nearly as abundant as ATP.
  • O-GlcNAc proteins are also Phosphoproteins
  • O-GlcNAc and Phosphorylation are often reciprocal.
  • Highly dynamic modification - a regulatory role.
  • ProteosomalProteinssWGA bound--GlcNAc ElutedUnbound-110-MW-20-pI 4pI 9pI 4pI 9Enrichment of O-GlcNAc Modified Proteins using sWGA:Silver stain of 80 ug total protein from each fraction(starting material 30 mg nucleocytoplamic lysate from HeLa cells)CTD 110.6 MAb Immunopurifies Many Glycoproteins From HeLa Cell Cytoplasmic and Nuclear Extract:2nd Dimension:10% SDS-PAGEVisualization:Silver StainpI: 3 --> 10Some Identified O-GlcNAc-Modified Proteins:Nuclear Pore Proteins - p62, Nup54, 155, 180, 153, 214, 358; Chromatin Proteins - ManyTranscription Factors - SP1, cfos, cJun, CTF, HNF1, v-ErbA, Pancreas Specific TF, SRF, c-Myc, p53, Estrogen Receptors, ß-catenin, NFKB, ELF-1, PAX-6, Enhancer Factor D, Human C1, Oct1, plakoglobin, YY1, PDX-1, CREB, Rb, p107, RNA Polymerase II.RNA Binding Proteins - HnRNP-G, Ewing Sarcoma RNA binding Protein, EF4A1, EF1alpha, 40S ribosomal s24, many ribosomal proteins.Phosphatases/Kinases/Adapters nuclear Tyr phos’ase p65, CKII, IRS-1,2, GSK3ß, PI3-kinaseCytoskeletal Proteins - cytokeratins 8, 13, 18, Neurofilaments H, M, L, Band 4.1, talin, vinculin, ankyrin, synapsin I, myosin, E-cadherin, cofilin, tau, Maps 2, 4, Dynein, alpha tubulin, AP3, AP180, ß-APP, ß-synuclein, piccolo.Chaperones - HSP27, alpha crystallins, HsC70, HsP70, HsP90 Metabolic Enzymes - eNOS, Enolase, Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, UDP-glucose synthase, glycogen synthaseOther Regulatory Proteins - Eukaryotic peptide chain initiation factor 2 p67, OGT, CRMP-2, Ubiquitin carboxy hydrolase (UCH), Glut 1, Annexin 1, Nucleophosmin, proteasome components C2, 5/9 and 9/14 of 26S proteasome regulatory & catalytic subunits, respectively, Q04323 UCH homolog, Sec23, Ran, peptidyl prolylisomerase, Rho GDP-dissociation inhibitor, GABA Receptor interacting protein 1, Viral Proteins - adenovirus fiber, SV40 Large T Ag;, Baculovirus Teg protein,Some Clues To O-GlcNAc Functions:(>~200 Papers Since 1984 Directly Concerned with O-GlcNAc)
  • Required for Life at Single Cell Level - (OGT -/+ still emb. lethal.)
  • Regulates Transcriptional Activation or Suppression, Dep. upon Metabolism - SP1, ERs, Stat5, NFkB, p53, c-Myc; Regulates Degradation.
  • Regulates Protein Synthesis (via p67-EIF2 kinase);(Gupta & Datta)
  • Regulates ß-catenin and E-cadherin trafficking (Andrews et al.)
  • Neurodegenerative Disease - Tau, ß-APP, NFs, O-GlcNAcase maps to late-AD locus; OGT Maps to Parkinson Dystonia Locus; O-GlcNAc is Reduced in Human AD.
  • YY1 Transcription Factor is Regulated by O-GlcNAc - Prevents binding to Rb.
  • O-GlcNAc on Glycogen Synthase Prevents its Activation by Insulin (Parker; McClain).
  • O-GlcNAc on eNOS prevents activation by Akt (Brownlee)
  • Retinoblastoma (Rb) is O-GlcNAc Modified in G1 & O-GlcNAc-Rb binds E2F.
  • O-GlcNAcylation of 26S Proteosome Inhibits Degradation; 5/19 and 9/14 of Catalytic Core and Reg. Core subunits, respectively, are modified (Cell 115, 715; BBRC 312,1284)
  • Regulator of Gibberellic Acid Signaling in Plants-OGT=Spy; Secret Agent
  • Blocks Insulin Signaling and OGT Over-Expression in Muscle or Adipose Causes Diabetes in Mice (McClain & Hanover)
  • Complex Interplay Between O-Phosphate & O-GlcNAc:OPTIONS FOR O-GlcNAc MODIFICATIONO-GlcNAc Transferase Summary:
  • OGT has been highly conserved throughout evolution.
  • OGT is a unique glycosyltransferase.
  • Localized near centromere on X-chromosome.
  • 1 Normal Allele is Required for ES Cell Viability (Marth et al.)
  • OGT structurecatalytic
  • Single gene encoding
  • 103 kDa peptide
  • migrates at 110 kDa
  • TPR domains - (tetratricopeptide repeats)Generates O-GlcNAc linkage on peptides Inexact peptide sequence motif for glycosylationAssociates with self and other proteins in complexLocated in nucleus and cytoplasmExpressed in all mammalian tissues studiedModified by O-GlcNAc and tyrosine-phosphateIdentified OGT-TPR Binding Proteins:(Proteins That Target OGT and Control Specificity)
  • Co-Repressor mSin3A binds OGT and Suppresses SP1 Driven Transcription (Kudlow)
  • GRIF-1 - Targets to GABA Receptor (Anne Stephenson) and Regulates its signaling; OGT binding protein (Sai Iyer).
  • Milton (Drosophila GRIF-1 analog) required for kinesin-mediated axonal transport of mitochondria to Synapses (Neuron 36, 1063 (Schwarz).
  • OIP106 - GRIF-1-Like Protein Targets OGT to RNA polymerase II - binds via TPRs.
  • Many more yet to be identified - pulled out by yeast 2H.
  • y = 0.6802 + 27.714x R= 0.99833 140012001000800600400200001020304050Effect of [UDP-GlcNAc] On OGT Activity In the Presence of Alkaline Phosphatase50 mM!!pmol incorporatedPhysiologicalRange[UDP-GlcNAc] mMO-GlcNAc’ase StructureSingle gene encodes 916 amino acid polypeptide of 103 kDa migrates at 130 kDaOGAOGA peptide cleaves GlcNAc from glycopeptidesPredominantly expressed in the cytoplasmExpressed in all human tissues studied Highly conserved in mammals and found in C. ElegansLocated on Chromosome 10 in humansFunctional aspects of O-GlcNAcase
  • Maps exactly to 10q23.1 - late onset Alzheimer’s Disease Locus.
  • Ogase has Histone Acetyltransferases activity.
  • Inhibition BLOCKS insulin signaling.
  • 53 nM Ki inhibitor ofO-GlcNAcase.Identification of O-GlcNAc Modified Proteins by Mass SpectrometryWhy Did O-GlcNAc Remain Undetected?
  • Generally Not Affect Gel Electrophoresis
  • Not Easily Labeled - No 32P!
  • Very Labile - both Chemically and Enzymatically- Falls Off in MS.
  • Stoichiometry Similar to O-Phosphate.
  • b ions - 164 251 348 449 536 633 720 848Y--S--P--T--S--P--S--K866 703 616 519 418 331 234 147 - y ionsO-GlcNAc (204)Fragmentation of GlcNAc-CTD by CID-MS/MS Parent ion 535 ([M+2H+] + 1 GlcNAc)NL 7.62e6y8866.310095Poor FragmentationNo Site Information.9085807570Peptide6560Relative Abundance5550sugar454035GlcNAc30y6410.3819.225203.8616.2b220b8433.815250.910848.4476.850200300400500600700800900100011001200m/zReplacement of O-GlcNAc with DTTUsing b-elimination/michael additionStrategy for O-GlcNAc/O-Phosphate site mapping100HDTTO-GlcNAcBAPPSVPVSerGSAPGRPSVPVSerGSAPGRPSVPVSerGSAPGRAlkaline inducedb-Elimination01314.3GlcNAcO(dehydroalanine)CH2CH216501100m/z(Serine-O-GlcNAc)CHSCH2CHOHCHOHCH2SHNHC100 (DTT)1247.4CNHCOO016501100m/z1001421.7DTT (or BAP)0HCH216501100m/zCNHCMALDI-TOFORelative intensityRelative intensityMichael Addition1. Provides tag forAffinity enrichment2. Tag is stable in massspectrometerRelative intensityBEMAD Useful for Simultaneous Mapping of O-GlcNAc and O-Phosphate:
  • O-GlcNAc much more sensitive to ß-Elimination.
  • Treatment with Phosphatase & O-GlcNAcase.
  • Density-Labeled DTT is Cheap & Available.
  • Same Approach work for ‘classical’ O-glycans.
  • Phosphorylation Mapping studies Must Account for Abundance of O-GlcNAc.
  • Another way to recognize O-GlcNAc proteinsMake GlcNAc derivative with a N3-reactive azide instead of AcUDP-GlcN -->-->Protein with O-GlcNProbe with FLAG or biotinDetect ProbeVocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR.Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9116-21Reciprocal O-GlcNAcylation & Phosphorylation of c-Myc:OHOHOThr58 is Mutation Hot Spot In Human Lymphomas.OHOAcNH-Leu-Leu-Pro-Thr58-Pro-Pro-Leu-O -O-P-O-O-Leu-Leu-Pro-Thr58-Pro-Pro-Leu-Mutations of c-Myc in LymphomasMajor O-GlcNAc Site.Site-Specific mAB - Thr58-O-GlcNAc:
  • Prevention of Phosphorylation at Ser 62 Elevates O-GlcNAc at Thr 58.
  • Stim. Of Growth Reduces O-GlcNAc at T58; Increases Phos. & vice versa.
  • Inhibition of GSK3ß increases O-GlcNAc at T58.
  • Regulates c-Myc association with Tumor Suppressor Rb p107.
  • GSK3ErkTransactivation DomainO-GlcNAcO-GlcNAcX XHeat ShockUVOsmotic StressFree RadicalsReductive StressHeat Shock ProteinsGlutathioneSODCatalaseThe rapid increase in O-GlcNAc and the effect of increased HSP70 in the presence of increased O-GlcNAc suggests that O-GlcNAc forms an integral component of the cell’s stress signaling pathways.Natasha ZacharaThe Addition of O-GlcNAc toProteins in Response to Stress is DynamicContTime post-heat shockCont11369244848 hWB: O-GlcNAc177114806450Cos-7 cells were treated with heat shock (45oC, 1h) and allowed to recover at 37oC.37O-GlcNAc Precedes HSP70 WB: HSC/HSP70O-GlcNAc is A Sensor of Cellular Stress:
  • O-GlcNAc and OGT are rapidly elevated in response to many forms of cellular stress
  • This is dose dependent; does not require mRNA or Protein Synthesis
  • Increased levels of O-GlcNAc results in increased thermotolerance
  • In part a result of more rapid increased HSP70 production/stabilization.
  • HSP70 as an O-GlcNAc lectin - 2 French Groups.
  • Decreased protein aggregation?
  • Decreased O-GlcNAc Results in decreased thermotolerance
  • UDP-GlcNAc as a Metabolic Sensor:Glucose MetabolismOverallEnergy Fatty AcidMetabolismUDP-GlcNAcNucleotide MetabolismNitrogenMetabolismREVIEW OF METABOLIC PATHWAYSElevation of O-GlcNAc Blocks Insulin Signaling:
  • Huge Literature - Diabetes Requires Glc to GlcNAc.
  • GlcN 10X more potent than Glc in inducing Insulin-Resistance.
  • O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.
  • Elevation of O-GlcNAc Blocks Insulin Signaling:
  • Huge Literature - Diabetes Requires Glc to GlcNAc.
  • GlcN 10X more potent than Glc in inducing Insulin-Resistance.
  • O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.
  • InsulinreceptorglucoseP-YP-YIRS1/2 p85Glut4vesiclep110HSPPKCz,dPI 3-kinasePDK1[UDP-GlcNAc]Thr 308-POGT!Insulin resistanceAKT1/2O-GlcNAcPUGNAcx-Ser/thr-x-xx-Ser/thr-x-xSer 9-PO-GlcNAcaseGSK3bGlycogen synthesis**?**Does Specific Elevation of O-GlcNAc Cause InsulinResistance? Yes!*2000010000 051%Insulin Resistance induced through the HBP correlates with increased O-GlcNAc in 3T3-L1 AdipocytesDeoxyglucose uptake (insulin dependent 14[C] DPM)Requires Both GlcN & Insulinfor Insulin-Resistance& for Elevated O-GlcNAc.Glucosamine (mM) 0 0 5 5chronic insulin (nM) 0 1 1 0WB: anti-O-GlcNAcPUGNAc Elevates O-GlcNAc and Induces Insulin Resistance in 3T3-L1 adipocytes:Acute insulin (nM) 0 0 1 1 10 10PUGNAc - + - + - +WB:anti-O-GlcNAc110.6 antibodyDeoxyglucose uptake (insulin dependent 14[C] DPM)PUGNAc - + - + - + - + - + Acute Insulin (nM) 0.1 0.33 1 3.3 10(A)(B)39%53 nM Ki inhibitor ofO-GlcNAcase.23%Insulin concentration (nM)Insulin Signaling Pathway for Glut4 Translocation to Plasma Membrane - Where is O-GlcNAc Blocking?-O-GlcNAc-O-GlcNAcMany O-GlcNAcIncluding Munc18-O-GlcNAc-->Glycogen SynthasePUGNAc does not effect the protein levels or insulin stimulated tyrosine phosphorylation of the insulin receptor or IRS-2.PUGNAC Blocks Insulin-Stimulated Phosphorylation of Threonine 308 on AKT/PkB*:AKT Activation is Blocked!PUGNAc Blocks Insulin-Stimulated Phosphorylation of GSK3ß at Ser9, But Not Thr202/204 MAPK or Ser473 on Akt/PKB:GSK3ß Activation is Blocked!Elevation of O-GlcNAc Blocks Insulin Signaling:
  • Blocks AKT phos. at T308 and S9 on GSK3ß
  • Inhib. OGase greatly increases OG on ß-catenin and IRS1.
  • InsulinreceptorglucoseP-YP-YIRS1/2 p85Glut4vesiclep110HSPPKCz,dPI 3-kinasePDK1[UDP-GlcNAc]Thr 308-POGT!Insulin resistanceAKT1/2O-GlcNAcPUGNAcx-Ser/thr-x-xx-Ser/thr-x-xSer 9-PO-GlcNAcaseGSK3bGlycogen synthesis?*?**Transgenic Mice with OverexpressedOGT in Muscle or Adipose - BecomeDiabetic. (McClain & Hanover)*Insulin Stimulates (10 min)Tyr-POf O-GlcNAc Transferase:Insulin (nM) 0 1 100 IP: OGTWB: OGTOGTOGT IP: OGTWB:PYNote: activity of these immunoprecipWas tested with no differences against CKII peptide. Post-translational modification may effectlocalization, substrate specificity, binding Partners, ect. (SH2 domain containing protein?)Phospho-tyrosine response would place OGT in Signaling pathway downstream of insulin Receptor. time point coincides with start of glucose Uptake assay. Fig. Insulin stimulated tyrosine phosphorylation of O-GlcNAc transferase (OGT). 3T3L1 adipocytes starved of growth factorsFor 16 hours were stimulated 10 minutes with insulin at suboptimal (1 nM) or optimal (100 nM) concentrations, and OGT Immunoprecipitates were western blotted for either OGT levels or phosphotyrosine. OGT is Directly in the IR Signaling Pathway.Proteomics Approach to O-GlcNAc & Insulin Signaling:Spot # response to insulin1 decreased O-GlcNAc2 decreased O-GlcNAc3 likely phosphorylation shift4 decreased O-GlcNAc5 likely phosphorylation shift6 decreased O-GlcNAc7 decreased O-GlcNAc456327unstimulated1pI 5pI 4.545632Insulin stimulated(100 nM for 10 min.)71(Small Portion of Gel Shown)Insulin Rapidly DecreasesO-GlcNAc on Many Proteins:Insulin Causes Major Changes in O-GlcNAc in 10 min.Fig. Insulin stimulates dynamic O-GlcNAcylation in 3T3L1 adipocytes. 3T3L1 adipocytes starved of growth factors for 16 hours Were stimulated with 100 nm insulin for 10 minutes, and whole cell lysates were separated by 2D gel electrophoresis and western blotted With an anti-O-GlcNAc specific antibody. Conclusions:
  • O-GlcNAc is a Major Regulatory PTM in all multicellular eukaryotes - Plants & Animals.
  • O-GlcNAc Accounts for Many of the Biological Affects Attributed Hexosamine Biosynthetic Pathway - Diabetes & Glucose Toxicity.
  • O-GlcNAc is Required for Life at the Single Cell Level.
  • O-GlcNAc is as abundant as Phosphorylation and Often Competes with it.
  • O-GlcNAc - “Metabolic Sensor” to Modulate Signaling & Transcription in Response to Cellular Status.
  • Many Toxic Effects of Hyperglycemia Likely Result From Dysregulation of the Balance Between O-GlcNAc and Phosphorylation.
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