1. ”Bittersweet
Roles of O-GlcNAc in Physiology
& Analytical Challenges”
Gerald W. Hart, Ph.D.
DeLamar Professor & Director
Department of Biological Chemistry
Johns Hopkins University
School of Medicine
725 N. Wolfe St., Baltimore, MD 21205-2185
Email: gwhart@jhmi.edu
Disclosures: Supported by NIH R01CA42486, R01DK61671; N01-HV-00240; P01HL107153, R24DK084949 and Dr. Hart is ”The
Beth W. and A. Ross Myers Scholar” of the Patrick C. Walsh Prostate Cancer Research Fund. Dr. Hart receives a share of
royalty received by the university on sales of the CTD 1 1 0. 6 antibody, which are managed by JHU.
Warren Symp. CCFC August 2012

2. O-GlcNAc is Different Than Other Glycans: Nucleocytoplasmic & Cycles
Outside
Plasma Membrane
O-GlcNAc Discovered
Early 1980s
Essentials of Glycobiology
Second Edition
Inside
Common classes of animal glycans

3. Detailed Glycomic Analysis of
O-GlcNAc Structural Diversity:
β
--Ser
(Thr)-
~3000 sites mapped to date

4. Properties of O-GlcNAc.
O-GlcNAc is Abundant on Nuclear & Cytosolic Proteins
2-5% Glucose
To Hexosamine
Biosynthesis
Pan >O-GlcNAc Antibody Western Blot - HeLa
UDP-GlcNAc
UDP
(Hart et al., Nature, April 2007)
 Highly Dynamic Enzymatic Modification of Ser and Thr residues by β-N-acetylglucosamine
 NOT elongated to more complex structures & Localized to the cytoplasm and nucleus.
 Present in all Metazoans studied, some bacteria & proteozoa, some fungi, plants & viruses.
 Highly abundant PTM (>~3000 identified proteins) & Often Reciprocal (Competitive) with
phosphorylation - Abundance = pancreas islets>>brain>>other tissues>liver.
 Dynamically cycling on Ser/Thr residues - Time scale & stoichiometry similar to phosphate.
 Cycling on proteins is Controlled by O-GlcNAc Transferase and by O-GlcNAcase

5. O-GlcNAc Has Extensive Crosstalk with Phosphorylation to Serve As
A Nutrient Sensor that Regulates Many Cellular Processes
Ann. Rev. Biochem. (2011)
UDP-GlcNAc
Is a Major
Node of Metabolism.
“O-GlcNAc or O-linked N-acetylglucosamine or hexosamine pathway” papers in PubMed = 1709 ~3000
proteins & 2500 Sites Mapped & Counting!

6. O-GlcNAc Regulates Many Cellular Processes:
“O-GlcNAc or O-linked N-acetylglucosamine or hexosamine pathway” papers in PubMed = 1737>
Essential Role in Lymphocyte Activation
- Both B- & T-lymphocytes; NFkB &
NFAT Nuclear Localization; EMBO J.
(2007) 26, 4368.
Regulates Protein Interactions
- Prevents YY1 binding Rb; Reg. ß-
Essential for Life - OGT is
required at the single cell, tissue and
organ level in plants and animals (Marth
et al.)
Polymerase II, SP1, CREB, NeuroD1,
PGC-1α, PDX-1, c-myc, p53, and mSin3A,
most transcription factors; Histones are
O-GlcNAcylated. OGT = Polycomb
gene.
Regulates the Proteasome &
Ubiquitination of p53 - Reduced
ATPase activity 19S cap;5/19 & 9/14 of
catalytic and core subunits, respectively
modified.
eg.eNOS;-diabetic erectile dysfunction;
glycogen synthase; RNA Pol II; ER, SV-40
Large T, c-myc,p53; Cancer
al.; Cole et al., Song et al.
Regulates Translation - eg.
Diabetes - Elevated O-GlcNAc blocks
Regulates Transcription - RNA
Blocks Phosphorylation -
Role in Neuronal Plasticity &
Synaptic Vesicle Trafficking &
Axonal Branching- Vosseller et
2-5% Glucose To
Hexosamine
Biosynthesis
catenin & E-cadherin trafficking
(Andrews et al.)
insulin signaling; Hyper-O-GlcNAc of
transcription factors important to glucose
toxicity; O-GlcNAc on NeuroD1 and PDX-1
regulates insulin transcription.
1,~3000 Proteins & 2500 Sites Mapped & Counting!
p67 binding to EIF2 kinase (Gupta)
Ribosome Proteins; mTOR pathway
(Hart et al., Nature, April 2007)
Regulates Histone Methylation
MLL5 GlcNAcylation triggers cell lineage
determination of HL60 through activation of
its HKMT activity. Nature April 2009
-
O-GlcNAc on SP1 Regulates HIV-1
latency and activation, and links viral
replication to the glucose metabolism
of the host cell. J. Virol. (March 2009)
Neurodegeneration - OGase Maps to
AD and OGT Parkinsons Loci; Most
proteins linked are O-GlcNAcylated; OGlcNAc reduced on tau etc. in AD; OGlcNAc reduced on key synaptic proteins.
Regulates Growth Hormone
(Gibberillic Acid) in Plants) Spy and Secret Agent are both OGTs.
Short-Term, Protects Cells From
Stress - Protects against multiple
forms of cellular stress; Protects
cardiac function after trauma and
ischemia.
Regulates Cell Cycle & Cytokinesis Affects Rate of Transition through cellcycle; Elevated OGT causes Polyploidy by
blocking cytokinesis - transient cytokinesis
complex.

7. Recent Mass Spectrometric Methods:
Chemico- enzymatic Enrichment combined
With Electron Transfer Dissociation (ETD)
Crosstalk Between GlcNAcylation &
Phosphorylation
is Surprisingly Extensive!

8. a. Competitive occupancy at same site
G
P
c-myc
1
58
439
OR
58
1
HLH
TAD
439
HLH
TAD
b. Reciprocal occupancy at different sites
P
C/EBPβ
G
P
P
179 180 181 184 189
1
RD
TAD
OR
345
179 180 181 184 189
1
RD
TAD
DBD
G
345
DBD
c. Simultaneous occupancy at different sites
P
IRS-1
P
307
1
PHD
PTBD
G
P
632 635
914
G
G
G
1009 1036 1041
1242
PRD
SRD
d. Site-dependent reciprocal or simultaneous occupancy
G
CaMKIV
1
57/58
AD
P
G
189 200
PKD
473
CaMBD
OR
1
57/58
AD
189 200
PKD
473
CaMBD

9. Simple Question:
In the absence of other stimuli,
how Does a Short Term (~2.5 hours)
Elevation (~3 fold) in Global
O-GlcNAcylation (caused by inhibiting O-GlcNAcase)
Affect Site Specific Phosphorylation?

10. Exp. Design: Affect on Phosphorylation if O-GlcNAc is Globally Increased?:
Okadaic Acid
OGase Inhibitors
Both Inhibitors
Quantitative proteomic
approach to delineate
global interplay
between
phosphorylation and
O-GlcNAcylation. A.
Flow chart for
phosphorylation
detection and sitespecific quantitation. B.
Protein level quantitation
using iTRAQ. C.
Formula used to
A.
IMAC at
Polypeptide level
calculate ROR
Titanium Dioxide
C.
=
[P-peptide] (B)
[Protein] (B)
[P-peptide] (A)
=
[P-peptide] (A)
[Protein] (A)
B. iTRAQ
ROR (A/B)
[Protein] (B)
[P-peptide] (B)
X
[Protein] (A)
1
=
ROR = Relative Occupancy Ratio
Proceedings Natl. Acad. Sci. (USA) (2008) 105 ,13793–13798.
Ratio [1] X
Ratio [2]

11. Site-Specific Phosphorylation Dynamics: Selected Examples
C
OA
P/N
Both
Cycling Site –
OA increases
P/N decreases
ATP-citrate synthase
R.TASFSESR.A
C
Site is Not
Cycling.
P/N increases global O-GlcNAc about
2-3 fold in 3hours
OA
P/N
Both
Insulin receptor substrate 2 (IRS-2)
R.VASPTSGLK.R
*All Data is Normalized to Spiked Internal STDs - 32 phosphorylation sites

12. Effects of a ~2-3-fold increase in Global O-GlcNAc for 2.5h:
Summary of ~700 Phosphorylation Sites Identified and Quantified:
Effects of Inhibiting
O-GlcNAcase on P Site
Occupancy:
~148 increased by P/N
~280 Sites Not Affected
by 3h inhibitor
~280 decreased by P/N
Nearly Every
Phosphorylation Site
That is Actively
Cycling is Affected by
GlcNAcylation!
Zihao Wang
Proceedings Natl. Acad. Sci. (USA) (2008) 105 ,13793–13798.

13. Kinases Are Regulated by
O-GlcNAcylation.

14. CTD110.6 (1:5000) – O-GlcNAcylation of Yeast Kinase (human) Chip
Print Array  OGT + UDP-GlcNAc  Immunoblot + Fluor 2ndary Ab
No OGT
Printed in Identical Pair Spots
+ OGT
Guanghui Han (collaboration Heng Zhu’s Lab.)

15. Some O-GlcNAcylated Kinases I.D. so far:
ERK-5
MAP2K3
p38
MAP3K14
MAPK1
MAP2K6
MAP3K6
CDK2
CDK5
CCRK
Polo-like K1
AURKB
PCTAIRE K1
pim-1 oncogene
src
PKCζ
PKCalpha
AKT
PIK3C3
TANK-binding K1
casein kinase 2*
PRKD2
C20orf97
CAMKK2 beta
CAMK IV
CAMK1G
CAMKII
Ser/thr
Ser/thr
Ser/thr
Ser/thr
Ser/thr
K16
K24
K25
K17B
K33
S6K1
PKLR
PDK3
PTK9
PAK4
BRD3
APEG1
SNARK
STE20-like kinase
AP2 associated K1
Heat Shock 27 p8
PACE-1
SFRS K1
RIO K2
RIO K3
LATS1
MATK
TRIB2
NEK8
FGR
CDK9
FLJ23356
ACUR2B
RIPK2
PNCK
ALS2CR2
CAMK2A
STK16
BMPRIB
PRKC1
CSNK1G2
PRKX
CDC2
STK4
CSNK2A1
CAMKK1
CSNK1E
HIPK1
ITK
PRK2
URK1
MAPK8
TESK2
CAMKK2
KSR2
ARK5
CAMKK2
TNK2
MAPK14
NUAK1
MAPK10
MYO3A
STK17A
PKN3
RP6-213H19.1
Dias et al. Biochemical and Biophysical Research Communications (2012)
46 O-GlcNAcylated Kinases in Synapse -”Disproportionately O-GlcNAcylated”
MCP Papers in Press. Published on May 29, 2012 (Burlingame) – i.d. 1750 O-GlcNAc sites in synapse.

16. O-GlcNAc at the Active Site Inhibits CAMKIV:
*Song et al. Cellular
Signaling (2007) O-GlcNAc
transferase is activated by
CaMKIV-dependent
phosphorylation under
potassium chloride-induced
depolarization in NG-108-15
cells

17. Relative Sizes of pT200 and og189 on CAMKIV
Surface models of N-acetylglucosamine (left foreground) and inorganic phosphate (right foreground), along with a cartoon model of the kinase
domain from human wild-type CaMKIV (center background) modeled from an X-ray crystal structure of human CaMKIγ. The amino acid residues
colored in green and red are those that are modified by GlcNAcylation and phosphorylation, respectively.

18. O-GlcNAc is a Negatively Modulates - Preventing Activation of CAMKIV
CaMKIV Activates OGT
PT
OGT
137
Inactive
OGA
ATP Pocket
GS
GS
189
137
CaMKIV
(Basal State)
GS
A
GS
Stimulation
356
Ca2+ increase
GS
Ca2+
CaM
GS
*Song et al. Cellular Signaling (2007) O-GlcNAc transferase is
activated by CaMKIV-dependent phosphorylation under potassium
chloride-induced depolarization = O-GlcNAc-Phosphate Reg. Cycle.
*
Activation loop
CaMKIV
GS
T57/S58
Activation is a 2 Step Process:
1 . Removal of O- GlcNAc
2. Activation by Phosphorylation &
Ca/CM
189
GS
356
B
T57/S58
CaMKK
137
200
PT
GS
CaMKIV
GS
C
GS
Active
356
(Stimulated State)
T57/S58
Dias et al. (2009) J. Biol. Chem. 284, 21327–21337
J. Cell Sci. 1 23, 13-22.

19. DYNAMIC CROSSTALK BETWEEN TWO ESSENTIAL NUTRIENT-SENSITIVE
ENZYMES:
O-GlcNAc Transferase (OGT) and AMPK-activated protein kinase (AMPK)
HIGHLIGHTS
1) AMPK regulates nutrient-sensitive nuclear localization of
OGT in myotubes, affecting global O-GlcNAcylation of
nuclear protein and K9 acetylation on histone 3.
2) AMPK phosphorylates Thr-444 on OGT, a residue that
regulates nuclear localization of OGT in response to
AMPK activation in myotubes
3)
AMPK is extensively and dynamically O-GlcNAcylated
(alpha 1 and 2 subunits & gamma subunits)
4) Acute global inhibition of O-GlcNAc cycling blunts
activation of AMPK
3000
John Bullen et al. submitted

20. Intein Chemical Ligation to Produce P344 & G347 CK2 Isoforms
Tarrant et al.
Nature Chemical Biology
2012

21. Using a Large Protein Array As Substrates, The Different
Modified Forms of CK2 Have Different Substrate Specificities.
S-Adenyosyl homocystein lyase
Good Substrate
For O-GlcNAc
Isoform.
Nucleosomal Assembly Protein 1
Good Substrate
For Phospho
Isoform.
Pin1 Assoc.
Also Regulates
Tarrant et al.
Nature Chemical Biology
2012

22. CK2 Specificity Is, In Part,
Controlled by Its Modifications:
Tarrant et al.
Nature Chemical Biology
2012

23. Extensive Crosstalk Between GlcNAcylation
And Phosphorylation Regulates Cytokinesis

24. A.OGT
DNA
B.
OGT
DNA
Over-expression of OGT
causes polyploidy.
O-GlcNAc transferase is associated
with the spindle and midbody
F.
GFP
Sup
OGT
TP
Sup
TP
G.
Tubulin
O-GlcNAc
Tubulin
O-GlcNAc
OGT
DNA
Purified spindles demonstrate
a ring of O-GlcNAc modified
proteins and localization with
tubulin near the spindle poles.
GFP
Sup
OGT
TP
Sup
O-GlcNAc modified proteins
are enriched at the midbody
and at the nascent nuclear envelope
GFP
H.
Sup
TP
250 kDa
250 kDa
160 kDa
105 kDa
75 kDa
75 kDa
50 kDa
50 kDa
TP
105 kDa
75 kDa
Sup
160 kDa
105 kDa
TP
250 kDa
160 kDa
OGT
50 kDa
Chad Slawson
IB: O-GlcNAc
O-GlcNAc is enriched
in the mitotic taxol pellet
and OGT over-expression
elevates protein GlcNAcylation.
IB: MPM-2
Mitotic phosphorylation
is disrupted after OGT
over-expression.
TP=taxol pellet
Coomassie Blue
OGT over-expression
alters protein expression
in the taxol pellet samples.
Molec. Biol. Cell 1 9, 41 30- 41 4

25. ETD is a Breakthrough for O-GlcNAc:
CAD
Synthetic C-Myc O-GlcNAc peptide
ETD
MS performed by Hunt et al. Univ. Virginia

26. The O-GlcNAc Ion Suppression Problem:
Ionization of O-GlcNAc
peptide is suppressed in
MALDI and ESI by naked
peptides
Solutions:
1. Top-down MS
1.
2.
High mass accurancy instrument
Need large amount pure protein.
Probably not effective for
complex samples
2. Enrich O-GlcNAc peptides
1.
2.
Direct enrichment of O-GlcNAc
peptides not effective
Enzymatic/chemical derivatization
available

27. Method to Quantify O-GlcNAc Site Occupancy:
Photocleavable Biotin Alkyne – 1 Step Synthesis
from Commercial Compounds:
O
O
C
O
NO2
O
C
O
N
O
C
N
H
+
H
H
S
N
CH
NH
HN
O
H3C
O
O
N
C
NH2
O
O
H2
C
C
Propylargylamine; (Aldrich)
NHS-ester reaction with primary amine; (Ambergen)
Olejnik et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7590-7594.
DMF, RT
O
C
H2
C C
CH
CH
NH
NO2
O
H
H
S
NH
O
H3C
O
HN
CH
C
N
O
O
C
N
H
O
Molecular and Cellular Proteomics (2010) 9: 1 53- 1 60.
N
C
O
Alkyne for
Click Chemistry

28. Photocleavable Biotin Tag for O-GlcNAc Site Mapping by ETD:
OH
OH
O
HO
HO
NH
O
O
HO
O
NHAC
O
O-GlcNAc-peptides
N
O
C
H3C
O
NH
HN
O
C
S
O
Attach to GalNAz-GlcNAc
by Click Chemistry
CH
H
N
C
O
C
N
H
O
N
N
NO2
H
N
H2
NH C
O
O
Affinity Purify - Streptavidin
Release by U.V cleavage
365 nm UV
OH
H3C
O
C
NH
HN
NO2
O
H
H
S
C
N
O
O
C
N
H
O
O
N
C
O
+
CO2
+
HO
HO
NH
O
Molecular and Cellular Proteomics (2010) 9: 1 53- 1 60.
N
Adds Pos. Charge
Recently: I.D. 274 O-GlcNAcylated proteins & Map 458 O-GlcNAc sites in Brain (Feng et al
PNAS in Press 2012)
H2
NH2 C
OH
O
N
N
O
HO
O
O
NHAC

29. Combinatorial Proteomic Approach to Identify Mitotic GlcNAcylation and Phosphorylation Sites
Method
GFP
The chemoenzymatic approach for O-GlcNAc enrichment
OGT
Click-Chemistry
SILAC labeling
Purify MB, digest w/
trypsin/Lys-C
UDP-GalNAz
GalT1, O/N
U.V.Cleavage
C18 trap
X-biotin-alkyne
Cycloaddition
Protein level quantification
SCX
Naked peptides
Better tag
technology
CID: Diagnostic Fragment Ions
FT
OH
FT
Avidin
chromatography
UV-cleavage
Quantification
Orbitrap-CID
Glyco-peptides
Site-mapping
FT-ETD
TiO2
chromatography
p-peptides
NH
elution
O
O
HO
O
O
NHAC
N
ID and quantification
NH2
H2
C
N
N
Orbitrap-CID
Spindle Midbody: Quantified: >700 Proteins;
>320 PhosphoSites by CID Orbitrap.
>~450
OH
O
HO
HO
1.
2.
3.
4.
5.
Easy release from avidin beads (w/o harsh condition)
Avoiding biotin fragmentation issue
Reduced precursor ion m/z
Adds Positive Chg for ETD; Small tryptics.
Diagnostic Fragment Ions: CID=peptide, not site.
GlcNAc Mass Pair:Peptides; High Res.Orbitrap CID): ~150 O-GlcNAc Sites by FT-ETD
Science Signaling 2010

30. Elevating OGT 2-Fold Dramatically Affects the CDK1 Circuit:
Aurora Kinase
Polo Kinase circuits
Also Affected in a
Similar Manner
Science Signaling 2010

31. DIBO-Alkyne based click chemistry for
the enrichment of O-GlcNAc peptides
Reactive group to –N3
Handle group
(biotin)
Linker arm
(Cleavable)
DIBO-Alkyne (Reagent 1)
•
•
•
•
Copper Free
No TBTA
Fewer steps
Reagent more stable and cheaper
Dr. Junfeng Ma

32. Conclusions:
♥ O-GlcNAc is a Major Regulatory Post-Translational Modification in all multicellular
eukaryotes - Plants & Animals & Viruses (some bacteria).
♥ O-GlcNAc is Required for Life at All Levels in Mammals and Plants.
♥ Crosstalk or Interplay Between O-GlcNAcylation & Phosphorylation is Extensive and
Involved in Many Cellular Processes.
♥ O-GlcNAc is Important to Transcription:Regulates Pol II & is Part of the Histone
Code where Some Sites are at Contact Regions with the DNA of the Nucleosome.
♥ O-GlcNAc - “Metabolic Sensor” That Modulates Signaling & Transcription in Response
to Cellular Status.
♥ Many Toxic Affects of Hyperglycemia Likely Result From Dysregulation of the
Balance Between O-GlcNAc and Phosphorylation = Glucose Toxicity.
♥ O-GlcNAcylation is Elevated in Nearly All Cancers Examined and it Likely Plays a
Role in Molecular Processes Leading to Cancer.
Disclosures: Supported by NIH R01CA42486, R01DK61671; N01-HV-00240; P01HL107153, R24DK084949 and Dr. Hart is ”The
Beth W. and A. Ross Myers Scholar” of the Patrick C. Walsh Prostate Cancer Research Fund. Dr. Hart receives a share of
royalty received by the university on sales of the CTD 1 1 0. 6 antibody, which are managed by JHU.

33. Acknowledgements
Hart Lab
Hunt Lab
Donald F. Hunt
Namrata D. Udeshi
Univ. Virginia
Genaro A. Ramirez-Correa, Weidong Gao,
and Anne M. Murphy
Department of Pediatrics/Division of Cardiology
Rick Huganair
For tools to study O- GlcNAc (eg. antibodies,
plasmids, protocols): email: gwhart@jhmi. edu
Brian Lewis, NIH