1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 15 The O-GlcNAc Modification Hud Freeze

2. “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 ADAPTED FROM

3. O-GlcNAc O- Linked N-Acetylglucosamine Tyr-Ser-Pro-Thr-Ser-Pro-Ser O AcNH HO OH O O AcNH HO OH O HO A dynamic post-translational modification O-GlcNAc Transferase O-GlcNAcase

4. Key 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. 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.

5. ProteosomalProteins

6. pI 4pI 9pI 4pI 9 Unbound sWGA bound--GlcNAc Eluted -110- MW -20- Silver stain of 80 ug total protein from each fraction (starting material 30 mg nucleocytoplamic lysate from HeLa cells) Enrichment of O-GlcNAc Modified Proteins using sWGA:

7. pI: 3 --> 10 2nd Dimension: 10% SDS-PAGE Visualization: Silver Stain CTD 110.6 MAb Immunopurifies Many Glycoproteins From HeLa Cell Cytoplasmic and Nuclear Extract:

8. Nuclear Pore Proteins - p62, Nup54, 155, 180, 153, 214, 358; Chromatin Proteins - Many Transcription Factors - Transcription 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-kinase Cytoskeletal 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 synthase Other 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 Identified O-GlcNAc-Modified Proteins:

9. Some Clues To O-GlcNAc Functions:  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) (>~200 Papers Since 1984 Directly Concerned with O-GlcNAc)

10. Complex Interplay Between O-Phosphate & O-GlcNAc:

11. OPTIONS FOR O-GlcNAc MODIFICATION

12. O-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.)

13. OGT structure catalytic TPR domains - (tetratricopeptide repeats) Generates O-GlcNAc linkage on peptides -Single gene encoding 103 kDa peptide migrates at 110 kDa Inexact peptide sequence motif for glycosylation Associates with self and other proteins in complex Expressed in all mammalian tissues studied Located in nucleus and cytoplasm Modified by O-GlcNAc and tyrosine-phosphate

15. Identified OGT-TPR Binding Proteins:  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. (Proteins That Target OGT and Control Specificity)

16. Effect of [UDP-GlcNAc] On OGT Activity In the Presence of Alkaline Phosphatase 0 200 400 600 800 1000 1200 1400 01020304050 y = 0.6802 + 27.714x R= 0.99833 pmol incorporated [UDP-GlcNAc] mM 50 mM!! PhysiologicalRange

17. O-GlcNAc’ase Structure OGA Expressed in all human tissues studied OGA peptide cleaves GlcNAc from glycopeptides Single gene encodes 916 amino acid polypeptide of 103 kDa migrates at 130 kDa Predominantly expressed in the cytoplasm Highly conserved in mammals and found in C. Elegans Located on Chromosome 10 in humans

18. Functional 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 of O-GlcNAcase.

19. Identification of O-GlcNAc Modified Proteins by Mass Spectrometry  Generally Not Affect Gel Electrophoresis  Not Easily Labeled - No 32 P!  Very Labile - both Chemically and Enzymatically- Falls Off in MS.  Stoichiometry Similar to O- Phosphate. Why Did O-GlcNAc Remain Undetected?

20. Fragmentation of GlcNAc-CTD by CID-MS/MS Parent ion 535 ([M+2H + ] + 1 GlcNAc) 200300400500600700800900100011001200 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 866.3 410.3 819.2 203.8 616.2 433.8 250.9 476.8 NL 7.62e6 Y--S--P--T--S--P--S--K b ions - 164251348449536633720848 866703616519418331234147 - y ions O-GlcNAc (204) 848.4 GlcNAc b2 y6 b8 y8 Poor Fragmentation No Site Information. Peptide sugar

21. Alkaline induced  Elimination C NHNH C O CH 2 (Serine-O-GlcNAc) (dehydroalanine) C NHNH C O CH 2 O GlcNAc H Strategy for O-GlcNAc/O-Phosphate site mapping 0 100 0 0 1314.3 PSVPVSerGSAPGR O-GlcNAc PSVPVSerGSAPGR DTT PSVPVSerGSAPGR BAP 1247.4 1421.7 m/z 1100 1650 m/z 1100 1650 m/z 1100 1650 MALDI-TOF Replacement of O-GlcNAc with DTT Using  -elimination/michael addition C NHNH C O CH 2 H DTT (or BAP) Michael Addition HSCH 2 CHOHCHOHCH 2 SH (DTT) 1. Provides tag for Affinity enrichment 2. Tag is stable in mass spectrometer Relative intensity

22. BEMAD 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.

23. Make GlcNAc derivative with a N 3 -reactive azide instead of Ac UDP-GlcN -->-->Protein with O-GlcN Probe with FLAG or biotin Detect Probe Another way to recognize O-GlcNAc proteins Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9116-21

24. OH O AcNH HO O -Leu-Leu-Pro-Thr 58 -Pro-Pro-Leu- O O Reciprocal O-GlcNAcylation & Phosphorylation of c-Myc: -P-O - -O-O-O-O Thr58 is Mutation Hot Spot In Human Lymphomas.

25. Mutations of c-Myc in Lymphomas Major O-GlcNAc Site. Transactivation Domain GSK3 Erk Site-Specific mAB - Thr58-O-GlcNAc: 1.Prevention of Phosphorylation at Ser 62 Elevates O- GlcNAc at Thr 58. 2.Stim. Of Growth Reduces O-GlcNAc at T58; Increases Phos. & vice versa. 3.Inhibition of GSK3ß increases O-GlcNAc at T58. 4.Regulates c-Myc association with Tumor Suppressor Rb p107.

26. Heat Shock UV Osmotic Stress Free Radicals Reductive Stress Heat Shock Proteins Glutathione SOD Catalase X O-GlcNAc The 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 Zachara

27. The Addition of O-GlcNAc to Proteins in Response to Stress is Dynamic Cos-7 cells were treated with heat shock (45 o C, 1h) and allowed to recover at 37 o C. 177 114 80 64 50 37 WB: O-GlcNAc WB: HSC/HSP70 1364848 h Time post-heat shockCont 924 1 O-GlcNAc Precedes HSP70

28. O-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

29. UDP-GlcNAc as a Metabolic Sensor: UDP-GlcNAc GlucoseMetabolism NitrogenMetabolism Fatty Acid Metabolism NucleotideMetabolism OverallEnergy

30. REVIEW OF METABOLIC PATHWAYS

31. Elevation of O-GlcNAc Blocks Insulin Signaling: Huge Literature - Diabetes Requires Glc to GlcNAc. Huge Literature - Diabetes Requires Glc to GlcNAc. GlcN 10X more potent than Glc in inducing Insulin-Resistance. GlcN 10X more potent than Glc in inducing Insulin-Resistance. O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals. O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.

32. [UDP-GlcNAc] HSP OGT x-Ser/thr-x-x O-GlcNAc O-GlcNAcase glucose Glut4 vesicle PDK1 AKT1/2 Thr 308-P PKC  GSK3  P-Y IRS1/2 Insulin receptor P-Y p85 p110 PI 3-kinase Glycogen synthesis Ser 9-P PUGNAc ! Insulin resistance Elevation of O-GlcNAc Blocks Insulin Signaling: Huge Literature - Diabetes Requires Glc to GlcNAc. Huge Literature - Diabetes Requires Glc to GlcNAc. GlcN 10X more potent than Glc in inducing Insulin-Resistance. GlcN 10X more potent than Glc in inducing Insulin-Resistance. O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals. O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.* * *?*?*?*? Does Specific Elevation of O-GlcNAc Cause Insulin Resistance? Yes! * *

33. Glucosamine (mM) 0 0 5 5 chronic insulin (nM) 0 1 1 0 Deoxyglucose uptake (insulin dependent 14 [C] DPM) 20000 10000 0 51% WB: anti-O-GlcNAc Insulin Resistance induced through the HBP correlates with increased O-GlcNAc in 3T3-L1 Adipocytes Requires Both GlcN & Insulin for Insulin-Resistance & for Elevated O-GlcNAc.

34. (A) Insulin concentration (nM) 23% 39% Acute insulin (nM) 0 0 1 1 10 10 PUGNAc - + - + - + WB:anti-O-GlcNAc 110.6 antibody PUGNAc - + - + - + - + - + Acute Insulin (nM) 0.1 0.33 1 3.3 10 Deoxyglucose uptake (insulin dependent 14 [C] DPM) (B) PUGNAc Elevates O-GlcNAc and Induces Insulin Resistance in 3T3-L1 adipocytes: 53 nM Ki inhibitor of O-GlcNAcase.

35. Insulin Signaling Pathway for Glut4 Translocation to Plasma Membrane - Where is O-GlcNAc Blocking?-O-GlcNAc Many O-GlcNAc IncludingMunc18 Glycogen Synthase --> -O-GlcNAc -O-GlcNAc

36. PUGNAc 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!

37. [UDP-GlcNAc] HSP OGT x-Ser/thr-x-x O-GlcNAc O-GlcNAcase glucose Glut4 vesicle PDK1 AKT1/2 Thr 308-P PKC  GSK3  P-Y IRS1/2 Insulin receptor P-Y p85 p110 PI 3-kinase Glycogen synthesis Ser 9-P PUGNAc ! Insulin resistance Elevation of O-GlcNAc Blocks Insulin Signaling: Blocks AKT phos. at T308 and S9 on GSK3ßBlocks AKT phos. at T308 and S9 on GSK3ß Inhib. OGase greatly increases OG on ß-catenin and IRS1.Inhib. OGase greatly increases OG on ß-catenin and IRS1. Transgenic Mice with Overexpressed OGT in Muscle or Adipose - Become Diabetic. (McClain & Hanover) * * *?*?*?*? * ?

38. Insulin (nM) 0 1 100 IP: OGT WB: OGT IP: OGT WB:PY OGT Fig. Insulin stimulated tyrosine phosphorylation of O-GlcNAc transferase (OGT). 3T3L1 adipocytes starved of growth factors For 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. Note: activity of these immunoprecip Was tested with no differences against CKII peptide. Post-translational modification may effect localization, 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. Insulin Stimulates (10 min)Tyr-P Of O-GlcNAc Transferase: OGT is Directly in the IR Signaling Pathway.

39. 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. Spot # response to insulin 1 decreased O-GlcNAc 2 decreased O-GlcNAc 3 likely phosphorylation shift 4 decreased O-GlcNAc 5 likely phosphorylation shift 6 decreased O-GlcNAc 7 decreased O-GlcNAc 1 1 2 2 3 3 4 4 5 5 6 6 7 7 pI 4.5pI 5 unstimulated Insulin stimulated (100 nM for 10 min.) Proteomics Approach to O-GlcNAc & Insulin Signaling : Insulin Rapidly Decreases O-GlcNAc on Many Proteins: Insulin Causes Major Changes in O-GlcNAc in 10 min. (Small Portion of Gel Shown)

40. 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.