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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
O- Linked N-Acetylglucosamine
A dynamic post-translational modification
OH
OH
HO
HO
O
O
HO
AcNH
O
O
HO
AcNH
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
O-GlcNAc Transferase
O-GlcNAcase
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.
O-GlcNAc proteins are also Phosphoproteins

O-GlcNAc and Phosphorylation are often
reciprocal.

Highly dynamic modification - a regulatory
role.
Non-Capsid
Viral
Proteins
Cytoplasmic Tails
Cytoplasmic Face of
of Membrane Proteins
Vesicle Proteins
Cytoskeletal Proteins
Proteosomal
Proteins
Chromatin
Proteins
Parasite
Proteins
Nuclear Pore
Proteins
RNA Polymerase II
& Transcription Factors
Tumor Suppressors RNA Processing
Proteins
& Oncogenes
Cytosolic
Enzymes
Protein Translation
Regulatory Factors
Enrichment of O-GlcNAc
Modified Proteins using sWGA:
sWGA bound--GlcNAc Eluted
Unbound
-110-
MW
-20-
pI 4
pI 9
pI 4
Silver stain of 80 ug total protein from each fraction
(starting material 30 mg nucleocytoplamic lysate from HeLa cells)
pI 9
CTD 110.6 MAb Immunopurifies Many Glycoproteins
From HeLa Cell Cytoplasmic and Nuclear Extract:
2nd Dimension:
10% SDS-PAGE
Visualization:
Silver Stain
pI: 3 --> 10
Some Identified O-GlcNAc-Modified Proteins:
Nuclear Pore Proteins - p62, Nup54, 155, 180, 153, 214, 358; Chromatin Proteins - Many
Transcription Factors - SP1, cfos, cJun, CTF, HNF1, v-ErbA, Pancreas Specific TF, SRF, cMyc, 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 GDPdissociation 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:
OH
HO
HO
O
O
HNAc
Site
A
Site
B
OH
HO
HO
O
O
OPO3-
HNAc
1
OPO3-
Site
A
Site
B
2
4
Site
A
Site
B
5
3
OH
HO
HO
O
O
HNAc
Site
A
Site
B
6
Site
A
7
Site
B
OPTIONS FOR O-GlcNAc MODIFICATION
QuickTime™ and a
GIF decompressor
are needed to see this picture.
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
et al.)
(Marth
OGT structure
catalytic
-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 glycosylation
Associates with self and other proteins in complex
Located in nucleus and cytoplasm
Expressed in all mammalian tissues studied
Modified by O-GlcNAc and tyrosine-phosphate
OGT mRNA
TPR Interacting
Effector Proteins
RNA Processing
Transcriptional
Regulation
Post-translational
Modifications
O-GlcNAc
TPR
Domain
OGT Splice
Variants
O-GlcNAc
Catalytic
Domain
Substrate
Selectivity
Proteolytic
Processing
OGT Variant
[UDP-GlcNAc]
[UDP]
Multimerization
O-GlcNAc
O-GlcNAc O-GlcNAc O-GlcNAc
OGT
O-GlcNAc Modified Proteins
O-GlcNAc
Identified 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
signaling; OGT binding protein (Sai Iyer).
(Anne Stephenson) and Regulates its
 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.
Effect of [UDP-GlcNAc] On OGT Activity
In the Presence of Alkaline Phosphatase
y = 0.6802 + 27.714x R= 0.99833
1400
50 mM!!
pmol incorporated
1200
1000
800
600
Physiological
Range
400
200
0
0
10
20
30
[UDP-GlcNAc] mM
40
50
O-GlcNAc’ase Structure
OGA
Single gene encodes 916 amino acid
polypeptide of 103 kDa
migrates at 130 kDa
OGA peptide cleaves GlcNAc from glycopeptides
Predominantly expressed in the cytoplasm
Expressed in all human tissues studied
Highly conserved in mammals and found in C. Elegans
Located on Chromosome 10 in humans
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.
Identification of O-GlcNAc
Modified
Proteins by Mass Spectrometry
Why 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 OPhosphate.
Fragmentation of GlcNAc-CTD by CID-MS/MS
Parent ion 535 ([M+2H+] + 1 GlcNAc)
866.3 y8
b ions - 164 251 348 449 536 633 720 848
Y--S--P--T--S--P--S--K
NL 7.62e6
Poor Fragmentation
866 703 616 519 418 331 234 147 - y ions
O-GlcNAc (204)
Relative Abundance
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
No Site Information.
Peptide
sugar
GlcNAc
203.8
b2
250.9
410.3
y6
616.2
819.2
433.8
b8
848.4
476.8
200
300
400
500
600
700
m/z
800
900
1000
1100
1200
Strategy for O-GlcNAc/O-Phosphate site mapping Replacement of O-GlcNAc with DTT
Using b-elimination/michael addition
Alkaline
induced
b-Elimination
GlcNAc
O
H
N
H
CH2
CH2
C
N
H
C
C
Relative intensity
O
(Serine-O-GlcNAc)
HSCH2CHOHCHOHCH2SH
(DTT)
1. Provides tag for
Affinity enrichment
DTT (or BAP)
N
H
0
100
1100
1650
m/z
1247.4
DTT
PSVPVSerGSAPGR
Relative intensity
Michael Addition
CH2
C
O-GlcNAc
PSVPVSerGSAPGR
C
(dehydroalanine)
O
H
1314.3
100
C
0
1100
100
1650
m/z
1421.7
2. Tag is stable in mass
spectrometer
BAP
PSVPVSerGSAPGR
Relative intensity
0
O
1100
m/z
MALDI-TOF
1650
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.
Another way to recognize O-GlcNAc proteins
Make GlcNAc derivative with a N3-reactive azide instead of Ac
UDP-GlcN
-->-->Protein with O-GlcN
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Probe with FLAG or biotin
Detect Probe
Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR.
Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9116-21
Reciprocal O-GlcNAcylation & Phosphorylation of c-Myc:
OH
HO
O
O
HO
Thr58 is Mutation Hot Spot
In Human Lymphomas.
AcNH
-Leu-Leu-Pro-Thr58-Pro-Pro-LeuO
-O-P-O-
O
-Leu-Leu-Pro-Thr58-Pro-Pro-Leu-
Mutations of c-Myc in Lymphomas
Major O-GlcNAc Site.
1.
GSK3
2.
Erk
Transactivation Domain
3.
4.
Site-Specific mAB - Thr58-O-GlcNAc:
Prevention of Phosphorylation at Ser 62 Elevates OGlcNAc 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.
O-GlcNAc
O-GlcNAc
X
Heat Shock
Heat Shock Proteins
UV
Glutathione
Osmotic Stress
SOD
Free Radicals
Catalase
X
Reductive Stress
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
The Addition of O-GlcNAc to Proteins in
Response to Stress is Dynamic
Cont
1
177
1
Time post-heat shock
3
6
9
24
48
Cont
48 h
WB: O-GlcNAc
114
80
64
50
37
O-GlcNAc Precedes HSP70
WB: HSC/HSP70
Cos-7 cells were treated with heat shock (45oC, 1h) and allowed to recover at 37oC.
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
UDP-GlcNAc as a Metabolic Sensor:
Overall
Energy
Glucose
Metabolism
UDP-GlcNAc
Nucleotide
Metabolism
Nitrogen
Metabolism
Fatty Acid
Metabolism
REVIEW OF METABOLIC PATHWAYS
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.
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.
Insulin
receptor
glucose
IRS1/2
*?
HSP
*
p85
p110
PI 3-kinase
PKCz,d
PDK1
[UDP-GlcNAc]
OGT
O-GlcNAc
x-Ser/thr-x-x
x-Ser/thr-x-x
O-GlcNAcase
PUGNAc
!
Insulin resistance
*
Thr 308-P
AKT1/2
Does Specific Elevation of
O-GlcNAc Cause Insulin
Resistance? Yes!
*
Ser 9-P
GSK3b
Glycogen synthesis
Glut4
vesicle
Insulin Resistance induced through the HBP correlates
with increased O-GlcNAc in 3T3-L1 Adipocytes
18000
16000
20000
14000
12000
51%
10000
Series1
8000
10000
Requires Both GlcN & Insulin
for Insulin-Resistance
& for Elevated O-GlcNAc.
6000
4000
2000
0
0
Glucosamine (mM)1 0
chronic insulin (nM) 0
WB: anti-O-GlcNAc
2
0
1
3
54
1
5
0
PUGNAc Elevates O-GlcNAc and Induces
Insulin Resistance in 3T3-L1 adipocytes:
Acute insulin (nM) (A)
PUGNAc
0
-
0
+
1
-
1
+
10
-
10
+
WB:anti-O-GlcNAc
110.6 antibody
(B)
39%
53 nM Ki inhibitor of
O-GlcNAcase.
23%
PUGNAc
Acute Insulin (nM)
- +
- +
- +
- +
0.1
0.33
1
3.3
Insulin concentration (nM)
- +
10
Insulin Signaling Pathway for Glut4 Translocation to
Plasma Membrane - Where is O-GlcNAc Blocking?
IR
Glut4
IRS
PI3K
-O-GlcNAc
-O-GlcNAc
PKCz
???
Many O-GlcNAc
Including
Munc18
PDK1
AKT
GSK3
-->Glycogen Synthase
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!
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.
Insulin
receptor
glucose
IRS1/2
*?
HSP
?
p85
p110
PKCz,d
PI 3-kinase
PDK1
[UDP-GlcNAc]
OGT
O-GlcNAc
x-Ser/thr-x-x
x-Ser/thr-x-x
O-GlcNAcase
PUGNAc
!
Insulin resistance
Transgenic Mice with Overexpressed
OGT in Muscle or Adipose - Become
Diabetic. (McClain & Hanover)
*
Thr 308-P
AKT1/2
*
Ser 9-P
GSK3b
Glycogen synthesis
Glut4
vesicle
Insulin Stimulates (10 min)Tyr-P
Of O-GlcNAc Transferase:
Insulin (nM)
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.
0
1
100
IP: OGT
WB: OGT
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.
OGT is Directly in the IR Signaling Pathway.
Proteomics Approach to O-GlcNAc & Insulin Signaling:
Insulin Rapidly Decreases
O-GlcNAc on Many Proteins:
4
(Small Portion of
Gel Shown)
5
6
Spot #
1
2
3
3
2
7
1
unstimulated
4
5
pI 5
pI 4.5
6
7
response to insulin
decreased O-GlcNAc
decreased O-GlcNAc
likely phosphorylation
shift
decreased O-GlcNAc
likely phosphorylation
shift
decreased O-GlcNAc
decreased O-GlcNAc
4
5
6
2
Insulin stimulated
(100 nM for 10 min.)
1
3
7
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.