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)

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

4. 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

5. O-GlcNAc O-linked b-N-AcetylglucosamineOH 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

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

7. Silver Staining and Putative Identification of O-GlcNAcase
From a Cow Brain Purification 30 25 40 50 60 70 80 90
120
100
160
220 kD 1 2 3 4 5 6 7
Protein #
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

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

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

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

11. O-GlcNAcase A neutral, nucleocytoplasmic b-N-acetylglucosaminidase
Originally identified as a Menigioma Autoantigen (MGEA5)--multiple splice variants
May have hyaluronidase activity (???)
Enzyme is a Substrate for the Executioner Protease of Apoptosis, Caspase-3
Recently demonstrated to be a Dual-Activity Enzyme w/ N-terminus having O-GlcNAcase Activity and C-terminus having Histone Acetyltransferase (HAT) Activity
PUGNAc--a GlcNAc analogue inhibitor (Ki~50nM)
10q24--Implicated in Multiple Neurological Disorders

12. 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

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

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

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

16. Classes of Proteins Modified By O-GlcNAc ~100 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

17. More Proteins and Many Sites to Go less than 60 sites mapped, some of the NP proteins have as many as 10 sites
pI 4
pI 9
Silver stain of anti-O-GlcNAc immunopurified proteins

18. 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

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

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

21. 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

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

23. FFA X Glucose Glutamine Glucosamine Uridine GFAT
Glc-6-P Fruc-6-P GlcN-6-P UDP-GlcNAc
GS OGT
Glycogen Glycolysis O-GlcNAc Modification Complex
Synthesis Glycosylation X
???
GSK3b
AKT
PDK Leptin
PI3-K
??? PKC IRS
Glut IR
Glucose Insulin