The OGlcNAc Modification

1
The O-GlcNAc Modification Chapter 14 author
Gerald Hart Lecturer Jamey Marth CMM-W
Bldg., Rm. 333 ph. 534-6526 For CD of class
request with mailing address to jmarth_at_ucsd.edu
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Chronology of the O-GlcNAc Linkage
O-GlcNAc linkage discovered in 1984 by Gerald Hart
The O-GlcNAc linkage was shown in 1986 to be
abundant in all subcellular organelles of rat
liver except mitochondria
O-GlcNAc found on polytene chromosomes of
Drosophila in 1989
Numerous metabolic regulatory proteins found
modified by O-GlcNAc (1986-2002)
O-GlcNAc Transferase (OGT) gene cloned in 1997
O-GlcNAc-ase (OGA) gene cloned in 2001
3
Structure of the O-GlcNAc Linkage
4
Early Method to Detect O-GlcNAc Linkage
5
Mapping O-GlcNAc Attachment Sites
6
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7
Reversible Intracellular Protein Modification by
GlcNAc and Phosphate
GlcNAc
Y
P
S
T
S
OGT
OGTase
Y
P
S
T
S
Y
P
S
T
S
PO4
kinase
phosphatase
Y
P
S
T
S
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Processes Associated with O-GlcNAc
Protein Interaction Nuclear Pore
Complex Crystallins binding with vinculin and
talin Synaptic vesicles binding to
cytoskeleton Neurofilament assembly Microtubule
function tau, beta-amyloid Transcription RNA
Pol II complex, Sp1 DNA binding p53 Viral
capsid envelopment
Protein Synthesis Blocking eIF-2 phosphorylation
Protein Turnover Estrogen receptor
Glucose Homeostasis Glucosamine in insulin
resistance PUGNAc activity in insulin
resistance
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Model of Transcriptional Regulation by O-GlcNAc
10
Model of O-GlcNAc in Alzheimers Disease
11
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
Highly conserved and found in C. Elegans
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OGA Structure
Single gene encodes 916 amino acid polypeptide
of 103 kDa migrates at 130 kDa
OGA
OGA peptide cleaves GlcNAc from glycopeptides
Predominantly expressed in the cytoplasm
Inhibited by GlcNAc, PUGNAc, but not GalNAc
Expressed in all human tissues studied
Highly conserved in mammals and found in C.
Elegans
Located on Chromosome 10 in humans
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Can a Model of OGT Deficiency Yield
Insight Regarding the Biological Role of this
Nuclear and Cytoplasmic Protein Modification?
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15
OGT Mutagenesis Strategies
1. Classical method
OGT gene
vector
Neo
OGT mutant
Neo
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OGT Mutagenesis Strategies
2. Conditional Mutagenesis
OGT gene
vector
loxP site
Neo
TK
OGT Parental mutant
Neo
TK
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OGT Alleles Following Cre Recombination
OGT parental mutant
n
e
o
t
k
Cre gancyclovir
OGT Null mutant
OGT Conditional mutant
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Only OGT Conditional Mutations are Found in
Embryonic Stem Cells
wt
wt
1
2
3
1
2
3
Cre
WT
2 loxP sites
OGT Parental Mutant
ES cell DNA
ES cell DNA
OGT Conditional mutant
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Deleting the OGT Gene Appears Lethal in ES Cells

D
N
A
-
D
N
A
OGT Null Mutant
24
48
144
24
hrs. post Cre recombination
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Production of Mice Bearing the OGT Conditional
Mutation
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Unusual OGT Gene Inheritance Pattern
M
F
M
F
F
M
M
M
wt
OGT Conditional Mutant
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Breeding of Female Mice Bearing the OGT
Conditional Mutation
M
F
x
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Segregation of the OGT Conditional
Mutant indicates an X-Linked Gene
Offspring
OGT Genotypes
Cond. Mutant only
Parental
wt only
Heterozygote
genotype
Sex
wt male
18
male
12
0
x
female
12
19
0
F/wt female
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The OGT Gene Resides on the Human X Chromosome
OGT FISH of Metaphase Spread
DAPI Stain
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Regional Localization of the OGT Gene
on Mouse and Human X Chromosomes
Human X
Mouse X
chromosome
chromosome
DXmit41
Xq13
DXmit95
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OGT mutagenesis in oocytes and allele segregation

Z
P
3
-
C
r
e
G
1
H
o
m
o
z
y
g
o
u
s
-
n
u
l
l
W
i
l
d
-
t
y
p
e

f
u
n
c
t
i
o
n
H
e
t
e
r
o
z
y
g
o
u
s
-
n
u
l
l
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Mice inheriting the OGT Conditional Mutation
(F) are viable, those inheriting the OGT Null
Mutation do not Survive

Offspring
OGT Genotype

Parental
Sex

OGT genotype

wt/Y

F/Y

wt/wt

F/wt

F/F

?/Y, F/?,







wt/?, or ?/?
F/Y male
male

10

7

-

-

-

-
x
female

-

-

-

4

7

-
F/wt female
F/Y male
male

-

11

-

-

-

-
x
female

-

-

-

-

9

-
F/F female
wt/Y male
male

11

0

-

-

-

0
x
female

-

-

10

0

-

0
F/wt female
ZP3-Cre
F/Y male
male

8

0

-

-

-

0
x
F/wt female
female

-

-

-

9

0

0
ZP3-Cre
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OGT and the Reversible O-GlcNAc Modification
Provide a Means of Modulating Phosphate-Dependent
Signal Transduction and the Function of
Multiple Cellular Proteins
OGT and the O-GlcNAc Modification are
Essential for Cellular Viability and Mouse
Embryogenesis