1. Essentials of Glycobiology April 28th, 2008 Ajit Varki Lecture 10 Chapter 17 : Unique Forms of Nucleocytoplasmic Glycosylation Chapter 18 :The O-GlcNAc modification

2. All Cells Are Coated with “Glycans” Electron micrograph of a human lymphocyte (Ruthenium Red staining)

4. Major Glycan Classes in Vertebrate Cells

5. General Questions for Lecture 10 1.Why was the presence of glycans in the nuclear and cytosolic compartments missed for so long? 2.What is the most striking mechanistic difference between nucleocytoplasmic and cell surface glycosylation? 3.What biochemical criteria would you require to demonstrate a glycan attached to a nuclear or cytoplasmic protein? 4.What conventional glycosylation pathways have steps that occur on the cytoplasmic side of membranes and could be potential sources of nucleocytoplasmic glycans? 5.How would you demonstrate the presence of GAGs in the nuclear compartment? 6.Compare initiating glycosylation reactions on mucins, proteoglycans, glycogenin, and skp1. 7.O-GlcNAc is now known to be the commonest form of glycosylation in the cell. Why did it take so long to be discovered? What was the serendipity involved in the discovery? 8.O-GlcNAc is thought to compete with phosphorylation for the same or similar sites on nuclear or cytosolic glycoproteins. What are the similarities and differences between O- GlcNAcylation and phosphorylation? 9.How does O-GlcNAc act as a "metabolic sensor"? 10.Speculate as to how O-GlcNAc might contribute to 'glucose toxicity' in diabetes

6. Potential mechanisms for the glycosylation of cytoplasmic proteins

7. Glycogen Synthesis: Opposing models for self-glucosylation of glycogenin

8. Model for glycogen biosynthesis

9. Mechanism of glycosylation of Skp1 in the cytoplasm of Dictyostelium

10. Rho family proteins regulate receptor-mediated actin cytoskeleton perturbations and are targets for bacterial toxins.

11. Glycosylation of Rho proteins by toxins occurs at Thr-37 and this has a key role in GTP/GDP nucleotide exchange by these G proteins

12. O-GlcNAc - A Dynamic Protein Modification

13. 2-D gel analysis of immunoaffinity-isolated O-GlcNAc–modified proteins from nucleocytoplasmic extracts of HeLa cells

14. O-GlcNAcylated proteins occur in many cellular compartments

15. O-GlcNAcylated proteins belong to many different functional classes.

16. O-GlcNAc cycling is regulated by the concerted action of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGase).

17. O-GlcNAcylation: complex dynamic interplay with O-phosphorylation

18. Protein phosphatase 1 catalytic subunit (PP1c), a key enzyme that removes O- phosphate residues from important regulatory proteins, is in a complex with OGT

19. O-GlcNAc transferase (OGT) is regulated by multiple complex mechanisms: transcriptional regulation of expression, differential mRNA splicing, proteolytic processing, posttranslational modification, and multimerization with itself and other proteins

20. UDP-GlcNAc is the donor for O-GlcNAc transferase (OGT) and an ideal sensor of the metabolic status of the cell.

21. Elevating O-GlcNAc blocks insulin signaling at many points.

22. Torres, C. R. & Hart, G. W. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J. Biol. Chem. 1984. 259:3308 PMID: 6421821 Yang et al. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature. 2008. 451:964. PMID: 18288188

23. News Article Accompanying Yang et al.

24. General Questions for Lecture 10 1.Why was the presence of glycans in the nuclear and cytosolic compartments missed for so long? 2.What is the most striking mechanistic difference between nucleocytoplasmic and cell surface glycosylation? 3.What biochemical criteria would you require to demonstrate a glycan attached to a nuclear or cytoplasmic protein? 4.What conventional glycosylation pathways have steps that occur on the cytoplasmic side of membranes and could be potential sources of nucleocytoplasmic glycans? 5.How would you demonstrate the presence of GAGs in the nuclear compartment? 6.Compare initiating glycosylation reactions on mucins, proteoglycans, glycogenin, and skp1. 7.O-GlcNAc is now known to be the commonest form of glycosylation in the cell. Why did it take so long to be discovered? What was the serendipity involved in the discovery? 8.O-GlcNAc is thought to compete with phosphorylation for the same or similar sites on nuclear or cytosolic glycoproteins. What are the similarities and differences between O-GlcNAcylation and phosphorylation? 9.How does O-GlcNAc act as a "metabolic sensor"? 10.Speculate as to how O-GlcNAc might contribute to 'glucose toxicity' in diabetes