1 / 26

Nucleocytoplasmic Glycosylation: O-GlcNAc Modifications and Enzymes

Explore O-GlcNAc modifications in nucleocytoplasmic proteins, enzymes like O-GlcNAc Transferase and O-GlcNAcase, and their roles in cellular function. Understand the interplay between O-GlcNAcylation and phosphorylation, with implications in disease.

askinner
Download Presentation

Nucleocytoplasmic Glycosylation: O-GlcNAc Modifications and Enzymes

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related