The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth
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Transcript of The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth
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:[email protected]
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 chromosomesof Drosophila in 1989
Chronology of the O-GlcNAc Linkage
O-GlcNAc Transferase (OGT) gene cloned in 1997
O-GlcNAc-ase (OGA) gene cloned in 2001
Numerous metabolic regulatory proteins found modified by O-GlcNAc (1986-2002)
Structure of the O-GlcNAc Linkage
Early Method to Detect O-GlcNAc Linkage
Mapping O-GlcNAc Attachment Sites
Reversible Intracellular Protein Modification by GlcNAc and Phosphate
Y P S T S
Y P S T SY P S T S
Y P S T S
OGT OGTase
kinasephosphatase
PO4
GlcNAc
Processes Associated with O-GlcNAc
Protein Interaction: Nuclear Pore ComplexCrystallins: binding with vinculin and talinSynaptic vesicles: binding to cytoskeletonNeurofilament assemblyMicrotubule function: tau, beta-amyloidTranscription: RNA Pol II complex, Sp1DNA binding: p53Viral capsid envelopment
Protein Synthesis:Blocking eIF-2 phosphorylation
Glucose Homeostasis:Glucosamine in insulin resistance‘PUGNAc’ activity in insulin resistance
Protein Turnover:Estrogen receptor
Model of Transcriptional Regulation by O-GlcNAc
Model of O-GlcNAc in Alzheimer’s Disease
OGT structure
catalytic
TPR domains - (tetratricopeptide repeats)
Generates O-GlcNAc linkage on peptides
-Single gene encoding103 kDa peptidemigrates at 110 kDa
Inexact peptide sequence motif for glycosylation
Associates with self and other proteins in complex
Highly conserved and found in C. Elegans
Expressed in all mammalian tissues studied
Located in nucleus and cytoplasm
Modified by O-GlcNAc and tyrosine-phosphate
OGA Structure
OGA
Expressed in all human tissues studied
Inhibited by GlcNAc, PUGNAc, but not GalNAc
OGA peptide cleaves GlcNAc from glycopeptides
Single gene encodes 916 amino acid polypeptide of 103 kDa migrates at 130 kDa
Predominantly expressed in the cytoplasm
Highly conserved in mammals and found in C. Elegans
Located on Chromosome 10 in humans
Can a Model of OGT Deficiency Yield InsightRegarding the Biological Role of this Nuclear
and Cytoplasmic Protein Modification?
OGT Mutagenesis Strategies
OGTgene
1. Classical method
vector
OGTmutant
Neo
Neo
OGT Mutagenesis Strategies
OGTgene
vector
OGTParentalmutant
2. Conditional Mutagenesis
Neo
Neo TK
TK
loxP site
OGT Alleles Following Cre Recombination
neo tk
+Cre+ gancyclovir
OGTConditionalmutant
OGT parentalmutant
OGTNullmutant
Only OGT Conditional Mutations are Found in Embryonic Stem Cells
wt wt1 12 23 3+Cre
OGTConditionalmutant
OGT Parental Mutant
WT
ES cellDNA
ES cellDNA
2 loxP sites
- D N A + D N A
Deleting the OGT Gene Appears Lethal in ES Cells
OGTNullMutant
hrs. post Cre recombination
24 48 14424
Production of Mice Bearing the OGT Conditional Mutation
x
50%50%
=
wt
OGTConditionalMutant
Unusual OGT Gene Inheritance Pattern
x
Breeding of Female Mice Bearing the OGT Conditional Mutation
50%25% 25%
=
Segregation of the OGT Conditional Mutantindicates an X-Linked Gene
Parental
genotype
Offspring
Sex
wt only ‘Heterozygote’Cond. Mutant only
wt male
x
F/wt female female
male 12
12 19
180
0
OGT Genotypes
The OGT Gene Resides on the Human X Chromosome
OGT FISH of Metaphase Spread DAPI Stain
DXmit41
DXmit95
Xq13
Mouse Xchromosome
Human Xchromosome
Regional Localization of the OGT Geneon Mouse and Human X Chromosomes
Heterozygous-null
G1
Homozygous-null
ZP3-Cre
Wild-type function
OGT mutagenesis in oocytes and allele segregation
G2
wt/Y malex
F/wt femaleZP3-Cre
F/Y malex
F/wt femaleZP3-Cre
Parental OGT genotype
Offspring
SexOGT Genotype
F/Y malex
F/F female
F/Y malex
F/wt femalemale 10 7 - - - -female - - - 4 7 -
wt/Y F/Y wt/wt F/wt F/F ∆/Y, F/∆, wt/∆, or ∆/∆
male - 11 - - - -female - - - - 9 -
male 11 0 - - - 0female - - 10 0 - 0
male 8 0 - - - 0female - - - 9 0 0
Mice inheriting the OGT Conditional Mutation (F) are viable, those inheriting the OGT Null Mutation do not Survive
OGT and the O-GlcNAc Modification are Essentialfor Cellular Viability and Mouse Embryogenesis
OGT and the Reversible O-GlcNAc Modification Provide a Means of Modulating Phosphate-Dependent
Signal Transduction and the Function of Multiple Cellular Proteins