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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: jmarth@ucsd.edu. Chronology of the O-GlcNAc Linkage. O-GlcNAc linkage discovered in 1984 by Gerald Hart.

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The O-GlcNAc Modification Chapter 14 author: Gerald Hart Lecturer: Jamey Marth

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  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@ucsd.edu

  2. 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. 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

  7. 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

  8. Model of Transcriptional Regulation by O-GlcNAc

  9. Model of O-GlcNAc in Alzheimer’s Disease

  10. 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

  11. 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

  12. Can a Model of OGT Deficiency Yield Insight Regarding the Biological Role of this Nuclear and Cytoplasmic Protein Modification?

  13. OGT Mutagenesis Strategies 1. Classical method OGT gene vector Neo OGT mutant Neo

  14. OGT Mutagenesis Strategies 2. Conditional Mutagenesis OGT gene vector loxP site Neo TK OGT Parental mutant Neo TK

  15. OGT Alleles Following Cre Recombination OGT parental mutant n e o t k +Cre + gancyclovir OGT Null mutant OGT Conditional mutant

  16. 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

  17. Deleting the OGT Gene Appears Lethal in ES Cells + D N A - D N A OGT Null Mutant 24 24 48 144 hrs. post Cre recombination

  18. M F x = M F 50% 50% Production of Mice Bearing the OGT Conditional Mutation

  19. Unusual OGT Gene Inheritance Pattern M F F F M M M M wt OGT Conditional Mutant

  20. 25% 50% 25% = M F Breeding of Female Mice Bearing the OGT Conditional Mutation M F x

  21. 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 19 female 12 0 F/wt female

  22. The OGT Gene Resides on the Human X Chromosome OGT FISH of Metaphase Spread DAPI Stain

  23. Regional Localization of the OGT Gene on Mouse and Human X Chromosomes Human X Mouse X chromosome chromosome DXmit41 Xq13 DXmit95

  24. G 2 OGT mutagenesis in oocytes and allele segregation Z P 3 - C r e G 1 W i l d - t y p e f u n c t i o n H o m o z y g o u s - n u l l H e t e r o z y g o u s - n u l l

  25. 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 female - - - 9 0 0 F/wt female ZP3-Cre

  26. 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

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