1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 16 NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION Hud Freeze

2. MAJOR FORMS OF CYTOPLASMIC GLYCOSYALTION Animals: Hyaluronan, Glycogen Plants: Cellulose Insects Yeast & Fungi: Chitin Traditional Glycobiology Studies ER- and Golgi-based Glycosylation

3. MITOCHONDRIAL GLYCOSYLATION FACT OR FICTION? SUGGESTIVE RESULTS FROM THE EARLY DAYS Suggested by radio labeling with sugar precursors Lectin binding studies SOME NAGGING PROBLEMS Contamination by other cellular components, esp ER How to get glycosyltransferases into mitochondria? How do substrates enter?

4. ER MITOCH CLOSE FRIENDS ??

5. ?

7. GLYCOSYLATION IN MITOCHONDRIA? Metabolic labeling with 35 S +/- deglycosylation 3 H-Mannose labeling

8. Specific mitochondrial protein complexes label with 3 H-Man The glycoprotein binds to The lectin ConA NADH-Ubiquinone-(Complex I) F 1 -ATPase (Complex-V)

9. Nuclear and Cytoplasmic Glycosylation In Favor Many papers Highly regarded journals Many Lectins Composition Metabolic Labeling Enzyme digestions Opposed Orientation of Transferases- Type II Membrane Proteins Nuclear Localization Signals No site mapping No structural analysis of glycan Purity

12. CYTOSOLIC GLYCOSYLATION

14. GN Tyr-194 GN Tyr-194 GN Tyr-194 1 2 3 4 GN Tyr-194 5 GN Tyr-194 Glycogenin is a Self Priming Glucosyl transferase UDP UDP-Glc Phosphorylase+ Debranching Enzyme UDP-Glc UDP GS Branching Enz+GS

15. Intersubunit Priming Tyr-194 Intrasubunit Priming Tyr-194

16. CYTOSOLIC GLYCOSYLATION

17. Stages of Dictyostelium Development

18. Skp1 glycosylation pathway in Dictyostelium Most complex form of cytoplasmic glycosylation

19. Verified Complex Cytoplasmic Glycosylation Only demonstrated for a single protein, SkpI, in Dictyostelium SkpI involved in ubiquitination of cell cycle proteins Attached O-linked chain at Pro-143 (not conserved) which is first hydroxylated to HyPro Gal -6Gal Fuc 1-2Gal 1-3-GlcNAc-HyPro GlcNAc and Fuc Transferase purified and shown to have very low Km for sugar nucleotides First Gal and Fuc are added by single transferase Mammalian homologs may not have this modification

20. CYTOPLASMIC GLYCOSYLATION

21. O-Mannose UDP-Glc Transfers Glc1-P to Mannose on a cystosolic protein 62kD protein is Phosphoglucomutase (Glc-1-P Glc-6-P) Transferase and phosphodiesterase found on many cells Membrane association regulated by modification with Glc-1-P Structure of underlying oligosaccharide not known

22. CYTOPLASMIC GLYCOSYLATION

24. CYTOSOLIC GLYCOSYLATION

25. PROTEOGLYCANS MADE IN THE NUCLEUS NO DELIVERY TO NUCLEUS? MAYBE MECHANISM? GAG SUBFRACTION IS HIGHLY ENRICHED IN ISOLATED NUCLEI SMALL FRACTION OF FGF FROM LYSOSOME MAY BE TRANSPORTED TO THE NUCLEUS

26. CYTOSOLIC GLYCOSYLATION

27. Ubiquitination of a New Form of -Synuclein by Parkin from Human Brain: Implications for Parkinson's Disease Sp22 is an O-linked glycosylated isoform of -synuclein. Failure of parkin to degrade it could cause some forms of PD SCIENCE, July 13, 2001

28. Accumulation of an O-glycosylated form of synuclein In autosomal recessive forms of Parkinsons disease? CYTOSOL E3-Ub-ligase

29. co-incubation with O-glycosidase and sialidase A shifted the 22-kD species to a 16-kD position (31), where it now co-migrated with the unmodified S monomer, Sp16, from crude brain extracts (Fig. 5C). We conclude from our data obtained by mass spectrometry analysis (21) and these enzymatic digestions that Sp22 is a posttranslationally modified form of human Sp16 containing O-linked sugars. 31. For enzymatic digestion of HP2A precipitates, N-glycosidase, sialidase A, endo-O- glycosidase and protein phosphatase-1 (Sigma) were used as per manufacturers' instructions. 21. The HP2A-specific 22-kD protein yielded tryptic peptides corresponding to aa 13-21, 44-58, 46-58, 59-80, 61-80, 81-96, and 81-97 of human Sp16 (GenBank accession # L08850), each ending with a lysine, as expected. Thr/Ser

30. Thr/Ser SIALIDASE Thr/Ser O-GLYCOSIDASE DIGESTION OF O-LINKED GalNAc sugar chain-- 2 easy steps 1. 2.

31. ARE THEY CORRECT OR NOT? WHATS YOUR OPINION?

33. Summary O-GlcNAc is only one form of cytoplasmic glycosylation Hyaluronan, glycogen, cellulose and chitin are examples Other forms of Cytoplasmic glycosylation exist Transferases and genes are characterized The roles of specific sugar chains in not known in most cases Lack of structural information is often a key deficiency Mitochondria may acquire N-glycosylated proteins from ER Other forms may exist, but proof requires structural analysis