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ESSENTIALS OF GLYCOBIOLOGY LECTURE 16 NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION

ESSENTIALS OF GLYCOBIOLOGY LECTURE 16 NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION Hud Freeze. MITOCHONDRIAL GLYCOSYLATION FACT OR FICTION?. SUGGESTIVE RESULTS FROM THE EARLY DAYS Suggested by radio labeling with sugar precursors Lectin binding studies SOME NAGGING PROBLEMS

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ESSENTIALS OF GLYCOBIOLOGY LECTURE 16 NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION

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

  2. 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?

  3. CLOSE FRIENDS ?? MITOCH ER

  4. ?

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

  6. Specific mitochondrial protein complexes label with 3H-Man F1-ATPase (Complex-V) NADH-Ubiquinone-(Complex I) The glycoprotein binds to The lectin ConA

  7. 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

  8. CYTOSOLIC GLYCOSYLATION

  9. CYTOSOLIC GLYCOSYLATION

  10. GN is Self Priming GS 3 GN GN 2 Tyr-194 Tyr-194 GS UDP UDP-Glc 1 UDP-Glc GN UDP Tyr-194 Phosporylase+ Debranching Enzyme 4 GS GN GS Tyr-194 GN Tyr-194 Branching Enz+GS 5

  11. Intersubunit Priming Tyr-194 Intrasubunit Priming Tyr-194 Tyr-194 Tyr-194

  12. CYTOSOLIC GLYCOSYLATION

  13. Stages of Dictyostelium Development

  14. Skp1 glycosylation pathway in Dictyostelium Most complex form of cytosolic glycosylation

  15. Verified Complex Cytosolic 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 • Gala-6GalaFuca1-2Galb1-3-GlcNA-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

  16. CYTOSOLIC GLYCOSYLATION

  17. 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

  18. CYTOSOLIC GLYCOSYLATION

  19. CYTOSOLIC GLYCOSYLATION

  20. PROTEOGLYCAN DELIVERY TO NUCLEUS? MAYBE KEY FINDING IS THAT A GAG SUBFRACTION IS HIGHLY ENTRICHED IN ISOLATED NUCLEI MECHANISM?

  21. CYTOSOLIC GLYCOSYLATION

  22. Ubiquitination of a New Form of a-Synuclein by Parkin from Human Brain: Implications for Parkinson's Disease SCIENCE, JUNE 28, 2001 aSp22 is an O-linked glycosylated isoform of a-synuclein. Failure of parkin to degrade it could cause some forms of PD

  23. Accumulation of an O-glycosylated form of a-synuclein In autosomal recessive forms of Parkinson’s disease? CYTOSOL

  24. 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 S containing O-linked sugars. a3 b3 a-Thr/Ser 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 S (GenBank accession # L08850), each ending with a lysine, as expected.

  25. DIGESTION OF O-LINKED GalNAc sugar chain-- 2 easy steps a3 b3 a-Thr/Ser 1. SIALIDASE b3 a-Thr/Ser 2. O-GLYCOSIDASE b3

  26. ARE THEY CORRECT OR NOT? WHAT’S YOUR OPINION?

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