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O-linked b -N-acetylglucosamine

O-linked b -N-acetylglucosamine. Nucleocytoplasmic Glycosylation O-GlcNAc Other Putative Nucleocytoplasmic Modifications In Dicty (Slime Mold and other diatoms) there is Gal-Gal-Fuc-Gal-GlcNAc-HyPro on a E3 Ubiquitin ligase subunit (Skp1, Chris West)

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O-linked b -N-acetylglucosamine

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  1. O-linked b-N-acetylglucosamine • Nucleocytoplasmic Glycosylation • O-GlcNAc • Other Putative Nucleocytoplasmic Modifications • In Dicty (Slime Mold and other diatoms) there is Gal-Gal-Fuc-Gal-GlcNAc-HyPro on a E3 Ubiquitin ligase subunit (Skp1, Chris West) • O-GalNAc?? A reported nucleocytoplasmic hexosaminidase D (N-acetylgalactosaminidase)

  2. The majority of the Gal-capped GlcNAc structures are O-linked Most are disaccharides !!! 1st Evidence for O-GlcNAc

  3. Effect of galactosylation on b-elimination of Lymphocytes: Intact Cells 22% Lysed Cells 72% Majority of O-GlcNAc Modified Proteins are intracellular??!!?? RL2 Coom Gal

  4. O-GlcNAcO-linked b-N-Acetylglucosamine OH OH HO HO O O O O HO HO NH NH O O CH3 CH3 Tyr-Ser-Pro-Thr-Ser-Pro-Thr O-GlcNAc Transferase 1998 O-GlcNAcase 2001

  5. O-GlcNAc Transferase (OGT) • 110 kDa Polypeptide (multiple splice variants) • Localized to the Nucleus and Cytoplasm • Functions as a Multimer • 11.5 TPR Repeats In N-Terminus • Several Binding Partners • No Obvious Recognition Motif at the Primary Sequence Level • Xq13-several neurological diseases map

  6. Protein # kD 1 220 2 160 120 3 100 90 4 80 5 70 60 6 50 7 40 30 25 Silver Staining and Putative Identification of O-GlcNAcase From a Cow Brain Purification Nanobore LC-ESI Mass Spectrometry Identification of Proteins (minimum of 5 Sequenced (MS/MS) Peptides) # ID 1. TATA binding protein-interacting-protein (TIP-120) 2. Putative O-GlcNAcase (similar to hyaluronidase) 3. Heat shock protein 110 4. Cullin 5. DNA K-type molecular chaperone hsc70 6. Amphiphysin & Calcineurin A 7. Dihydropyrimidinase related protein-2

  7. 1091.7 2744 100 80 a, CTD-GalNAc, Mock b, CTD-GalNAc + O-GlcNAcase 60 1091.7 2930 100 1069.7 40 Na+ 80 Na+ 20 60 0 0 % Intensity 830 894 958 1022 1086 1150 40 1069.7 20 0 0 830 894 958 1022 1086 1150 d, CTD-GlcNAc + O-GlcNAcase c, CTD-GlcNAc, Mock 866.6 1091.8 2100 100 5158 100 1069.8 Na+ 80 80 -203.2 1091.8 60 Na+ 60 % Intensity 40 40 1069.8 20 20 0 0 0 0 830 894 958 1022 1086 1150 830 894 958 1022 1086 1150 Mass (m/z) Mass (m/z) Cloned O-GlcNAcase is Specific for O-GlcNAc Peptides Mass (m/z)

  8. O-GlcNAcase O-GlcNAc Western Specific (110.6) Blot Western Blot 118 --> MW 38 --> 118 --> MW 61 --> Overexpression of O-GlcNAcase Lowers Global O-GlcNAc Levels

  9. 100 90 80 80 60 40 20 20 10 0 Cytosol Nucleus Cytosol Nucleus Localization of O-GlcNAcase Activity / mg Total Activity Fractional Activity Cell Fraction Fig. 5

  10. O-GlcNAcase • A neutral, nucleocytoplasmic b-N-acetylglucosaminidase • Originally identified as a Menigioma Autoantigen (MGEA5)--multiple splice variants • May have hyaluronidase and HAT activity (???) • Enzyme is a Substrate for the Executioner Protease of Apoptosis, Caspase-3 • PUGNAc--a GlcNAc analogue inhibitor (Ki~50nM) • 10q24--Implicated in Multiple Neurological Disorders • SNP in O-GlcNAcase strongly correlated with type II diabetes in Mexican-American populations

  11. Features of O-GlcNAc • NOT elongated • Nucleocytoplasmic Proteins • Dynamic & Inducible • Enzymes for its Addition (OGT) and Removal (O-GlcNAcase) are Nucleocytoplasmic • OGT Knock-Out is Lethal at the ES cell stage • X-linked OGT, Females Heterozygotes are NOT mosaic • Reciprocity with Phosphorylation (Yin-Yang) on Some Proteins

  12. O-GlcNAcylation Phosphorylation O-GlcNAcase S/T Kinase PROTEIN -O-PO3 -O-GlcNAc PROTEIN PROTEIN PROTEIN O-GlcNAc Transferase S/T Phosphatase

  13. OH O HO Thr58 is Mutation Hot Spot In Human Lymphomas. O HO AcNH -Leu-Leu-Pro-Thr58-Pro-Pro-Leu- O -P-O- -O O -Leu-Leu-Pro-Thr58-Pro-Pro-Leu- Reciprocal Glycosylation & Phosphorylation of c-Myc: J Biol Chem. 1995 Aug 11;270(32):18961-5.

  14. OGT(AL28) Western S/T Phosphatase (FL-18) Western 118 kDa 38 kDa PP PreBleed OGT F. Wash Bound PP-Ab OGT Microcystin Sepharose Purification IP Antibody IP Antibody Silver Stain Western 118 85 OGT 61 50 IgG 38 Phosphatase IgG Markers Yin-Yang Complex Extract Isolation of a Yin-Yang Complex J Biol Chem. 2004 Sep 10;279(37):38466-70.

  15. Classes of Proteins Modified By O-GlcNAc~200 Proteins Identified to Date • Cytoskeletal Components Tau, Vinculin • Hormone Receptors ERa&b • Kinases & Other Signaling Molecules CKII, eNOS, IRS-2 • Nuclear Pore Proteins NUP62, NUP155 • Oncogenes & Tumor Suppresors p53, Rb • Transcription Factors c-myc, Sp1, Pax6 • Metabolic Enzymes GAPDH, Pyruvate Kinase • Transcriptional and Translational Machinery RNA Pol II, EIF4A • Viral Proteins SV40 T Antigen, v-Erb-a • Heat-Shock Proteins HSP90, a-crystallin

  16. More Proteins and Many Sites to Goless than 90 sites mapped, some of the NP proteins have as many as 10 sites pI 9 pI 4 Silver stain of anti-O-GlcNAc immunopurified proteins

  17. Detection of O-GlcNAc • Antibodies (RL-2 & 110.6) • GalT labeling • Radiolabeled GlcN • GlcNAc-azido followed by Staudinger Rxn • Beta-elimination/Michael Addition • Pilot Approaches--Sulfotranferases and Kinases

  18. Modulation of O-GlcNAc Levels By A Variety of Stimuli 60min T-Cell 1-5 minute Neutrophil GlcN+INS Thermal Activation Stimulation O/N Feeding Stress Hart Marchase Hart Hart

  19. Acute insulin (nM) 0 0 1 1 PUGNAc - + - + WB:anti-O-GlcNAc 110.6 antibody A Role for O-GlcNAc in Insulin Resistance (the Hallmark of Type II Diabetes) Insulin Resistance Insulin-Stimulated Glucose Uptake In Adipocytes Is Impaired By PUGNAc

  20. 1 2 3 4 5 1: No Serum (NS) 2:Serum 3:NS+P 4: NS+I 5. NS+P+I Role for O-GlcNAc in Modulating Anti-Apoptotic Action of Insulin A. B. PUGNAc Inhibits Insulin’s Anti-Apoptotic Action in R28 Retinal Neurons & CHO-IR Cells FITC-Annexin V and PI DNA fragmentation assay.

  21. * * ogt wt oga * p<0.01 ogt-1 WT oga-1 Mark O-GlcNAc Western Blot Deletions in the O-GlcNAc Cycling Enzymes Modulate Median Lifespan in C. elegans anti-alpha-tubulin www.desc.med.vu.nl/ NL-taxi/ICE/C_elegans1.jpg

  22. Functions Postulated for O-GlcNAc • Blocking Phosphorylation Sites (CTD of RNA Pol II, Tau) • Regulation of Chromatin Structure (msin3A) • Regulation of Transcriptional Activation (Sp1) • Regulation of Translational Initiation (gp67-eIF2A) • Regulation of Protein:Protein Interactions (STAT5-CBP) • Regulation of Insulin Signal Transduction • (At or Upstream of Akt) • Regulation of Adipocytokine Secretion (Transcriptional?) • Protection from Stress (Upregulation of HSP?) • Apoptosis (by inhibition of Akt activiation?) • GENERAL THEME??? • Glucose Sensor and Regulator of Energy Homeostasis

  23. Glucose Metabolism High-Energy Metabolism Amino Acid Metabolism UDP-GlcNAc Nucleotide Metabolism Fatty Acid Metabolism

  24. FFA Glucose Glutamine Glucosamine Uridine GFAT Glc-6-P Fruc-6-P GlcN-6-P UDP-GlcNAc GSOGT Glycogen GlycolysisO-GlcNAc ModificationComplex Synthesis Glycosylation X ??? GSK3b AKT PDK-1 Leptin PI3-K ??? PKC IRS Glut4 IR Glucose Insulin

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