the o-glcnac modification chapter 14 author: gerald hart lecturer: jamey marth
DESCRIPTION
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]. Chronology of the O-GlcNAc Linkage. O-GlcNAc linkage discovered in 1984 by Gerald Hart. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/1.jpg)
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]
![Page 2: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/2.jpg)
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)
![Page 3: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/3.jpg)
Structure of the O-GlcNAc Linkage
![Page 4: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/4.jpg)
Early Method to Detect O-GlcNAc Linkage
![Page 5: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/5.jpg)
Mapping O-GlcNAc Attachment Sites
![Page 6: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/6.jpg)
![Page 7: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/7.jpg)
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
![Page 8: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/8.jpg)
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
![Page 9: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/9.jpg)
Model of Transcriptional Regulation by O-GlcNAc
![Page 10: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/10.jpg)
Model of O-GlcNAc in Alzheimer’s Disease
![Page 11: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/11.jpg)
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
![Page 12: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/12.jpg)
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
![Page 13: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/13.jpg)
Can a Model of OGT Deficiency Yield InsightRegarding the Biological Role of this Nuclear
and Cytoplasmic Protein Modification?
![Page 14: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/14.jpg)
![Page 15: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/15.jpg)
OGT Mutagenesis Strategies
OGTgene
1. Classical method
vector
OGTmutant
Neo
Neo
![Page 16: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/16.jpg)
OGT Mutagenesis Strategies
OGTgene
vector
OGTParentalmutant
2. Conditional Mutagenesis
Neo
Neo TK
TK
loxP site
![Page 17: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/17.jpg)
OGT Alleles Following Cre Recombination
neo tk
+Cre+ gancyclovir
OGTConditionalmutant
OGT parentalmutant
OGTNullmutant
![Page 18: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/18.jpg)
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
![Page 19: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/19.jpg)
- D N A + D N A
Deleting the OGT Gene Appears Lethal in ES Cells
OGTNullMutant
hrs. post Cre recombination
24 48 14424
![Page 20: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/20.jpg)
Production of Mice Bearing the OGT Conditional Mutation
x
50%50%
=
![Page 21: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/21.jpg)
wt
OGTConditionalMutant
Unusual OGT Gene Inheritance Pattern
![Page 22: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/22.jpg)
x
Breeding of Female Mice Bearing the OGT Conditional Mutation
50%25% 25%
=
![Page 23: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/23.jpg)
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
![Page 24: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/24.jpg)
The OGT Gene Resides on the Human X Chromosome
OGT FISH of Metaphase Spread DAPI Stain
![Page 25: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/25.jpg)
DXmit41
DXmit95
Xq13
Mouse Xchromosome
Human Xchromosome
Regional Localization of the OGT Geneon Mouse and Human X Chromosomes
![Page 26: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/26.jpg)
Heterozygous-null
G1
Homozygous-null
ZP3-Cre
Wild-type function
OGT mutagenesis in oocytes and allele segregation
G2
![Page 27: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/27.jpg)
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
![Page 28: The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth](https://reader034.vdocuments.us/reader034/viewer/2022051620/56814363550346895dafde40/html5/thumbnails/28.jpg)
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