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@ucsd.edu

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