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O-Glycosylation

Glc. GlcNAc. GalNAc. Man. Fuc. Ser/Thr. Ser/Thr. Ser/Thr. Ser/Thr. Ser/Thr. O-Glycosylation. Notch Thrombospondin Factor IX. Yeast mannoproteins a -dystroglycan cadherins. Notch Coagulation Factors Fibrinolytic Factors. Nuclear Proteins Cytoplasmic Proteins SEPARATE LECTURE.

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O-Glycosylation

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  1. Glc GlcNAc GalNAc Man Fuc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr O-Glycosylation Notch Thrombospondin Factor IX Yeast mannoproteins a-dystroglycan cadherins Notch Coagulation Factors Fibrinolytic Factors Nuclear Proteins Cytoplasmic Proteins SEPARATE LECTURE Mucins ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation After Esko, J

  2. O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry) b-elimination O-Glycosidic Linkage GalNAc GalNAc a a Ser After Esko, J

  3. Glycan synthesis in a cellular context Most O-Glycosylated proteins are synthesized in the secretory pathway

  4. Glc GlcNAc GalNAc Man Fuc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr O-Glycosylation

  5. a3 b4 a3 b3 b4 b3 b4 a3 b3 b6 Ser/Thr Mucin-Type O-GalNAc Glycans • Major extracellular vertebrate O-glycan • Begins in cis-Golgi (ERGIC) by attachment of GalNAc in a-linkage to specific Ser/Thr residues • Assembly is simpler than N-linked chains - no lipid intermediate is used • Always involves nucleotide sugars • Always occurs by addition to non-reducing terminus or by branching After Esko, J

  6. Polypeptide GalNAc Transferases Regions in white, pink, red, and black represent, respectively, 0–29%, 30–69%, 70–99%, and 100% sequence identity (Hagen et al. (2003) Glycobiology 13:1R-16R). • >20 ppGalNAcT family members • Share structural features in active site • Some have lectin (ricin) domain After Esko, J

  7. T (TF) Antigen Core 1 GalT (cis) Core 2 GlcNAcT Tn Antigen ST6GalNAc1 (trans) b3 b6 b3 b3 a3 Sialyl Tn Antigen Disialyl T Antigen Ser/Thr Ser/Thr a6 a6 Ser/Thr b3 Ser/Thr Ser/Thr Ser/Thr Core 1 and Core 2 Synthesis After Esko, J

  8. From: Tongzhong Ju and Richard D. Cummings

  9. Ser/Thr Core 3 and Core 4 Synthesis Core 3 GlcNAcT Core 4 GlcNAcT b3 b3 b6 Ser/Thr Ser/Thr After Esko, J

  10. Core 1 Core 2 Core 3 Core 4 b3 b6 Ser/Thr Core 6? Core 7 Core 8 Core 5 b6 a6 a3 b3 a3 b3 b3 b6 Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Unusual Core O-Glycan Structures After Esko, J

  11. Mucins are Heavily O-glycosylated • Apomucin contain tandem repeats (8-169 amino acids) rich in proline, threonine, and serine (PTS domains) • Glycosylation constitutes as much as 80% of mass and tends to be clustered - bottle brush • Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts After Esko, J

  12. Mucin Production Lung Epithelium Goblet cells in intestinal crypts After Esko, J

  13. Mucins: Protective Barriers for Epithelial Cells • Lubrication for epithelial surfaces • Modulate infection: • Receptors for bacterial adhesins • Secreted mucins can act as decoys • Barrier against freezing: • Antifreeze glycoproteins • [Ala-Ala-Thr]n≤40 with Core 1 disaccharides After Esko, J

  14. Questions • What is the function of multiple polypeptide GalNAc transferases? • How is tissue specific expression of transferases regulated? • How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues? • What roles do chaperones play? After Esko, J

  15. Glc GlcNAc GalNAc Man Fuc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr O-Glycosylation

  16. O-Fuc • Two Flavors: Mono and Tetrasaccharide • One of the clearest examples of glycosylation (Fringe) modulating signal transduction • What other proteins carry O-Fuc and how does glycosylation modulate activity? • How is glycosylation regulated?

  17. Glc GlcNAc GalNAc Man Fuc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr O-Glycosylation

  18. O-Glc Pathway Shao, L. et al. Glycobiology 2002 12:763-770; doi:10.1093/glycob/cwf085

  19. Rumi is OGluT KDEL Retention Signal Temp. Sensitive Mutation

  20. O-Glc • Always a trisaccharide? • Glc & Xyl (except for proteoglycans) rarely used on mammalian glycoproteins--why both here? Many of the same proteins as O-Fuc modifed, why? • Role in Modulating Signaling? Regulated by enzymes or sugar nucleotide availability?

  21. Glc GlcNAc GalNAc Man Fuc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr O-Glycosylation

  22. POMT1 in the ER In Complex With POMT2 Uses Dol-Man As Donor

  23. Alpha-Dystroglycan

  24. Affected Biochemical Disease Species Biochemical Phenotype Gene Lesion Walker - Warburg O - Man addition to Decreased protein O - Human POMT1 Syndrome Ser/Thr mannosylation a Muscle - Eye - Addition of GlcNac Underglycosylated - DG, Human POMGnT1 b Brain D isease 2 to O - Man uncapped O - Man Fukuyama - type Glycosyltransferase - a Human Fukutin Underglycosylated - DG MDC like protein Fukutin - Limb - Girdle and Glycosyltransferase - a Human Related Underglycosylated - DG MDC 1C l ike Golgi protein Protein Myodystrophy, Mouse Glycosyltransferase - a myd LARGE Underglycosylated - DG like Golgi protein MDC 1D Human Muscular Dystrophies associated with glycosylation of a – DG (oversimplified) Other Disease causing genes: B4GAT1, POMGnT2, DPM1-3, ISPD, TMEM5 ….

  25. Mammalian O-Mannosylation POMGnT1 POMGnT1+GnTVb POMGnT2

  26. O-Man b-6 Branching in the Brain (primarily) GnTV-B Primarily Responsible

  27. A New Glycan Structure on a-DG that is Large-dependent Science (2010) 327:88-92

  28. The ECM binding O-Man Structure (also the receptor for certain clades of arenaviruses including Lassa) Praissman J L et al. eLife Sciences 2014;3:e03943

  29. +1, 1100 a-DG KO Brain MS/MS Analysis of the O-Man Classical Tetrasaccharide Glycan Structure Released from Brain Proteins Derived from the a-DG knock-out animal. Greyed out structures represent their loss.

  30. Other O-Mannosylated Proteins: • The Cadherin Family of Proteins • -also involved in adhesion • RPTPb • -Receptor protein tyrosine receptor • 3. OTHERS???

  31. O-Man • O-Man is clearly involved in CMD • What mammalian proteins (especially in the brain) are O-Man modified besides a-DG? • What are the functions of fukutin, ispd, TMEM5 in O-mannosylation? • Why the heterogeneity in O-Man structures, what specific structures at what sites on the protein modulate specific interactions? • What is relationship between O-Man and O-GalNAc?

  32. Glc GlcNAc GalNAc Man Fuc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr O-Glycosylation

  33. A few more O-glycans…… GlcNAc Xyl Gal Glc Man Fuc GalNAc b Ser/Thr Ser/Thr Ser/Thr Ser/Thr Hyl Ser Hyp Ser/Thr O-Xyl….precursor for GAGs… O-GlcNAc….separate lecture—note: recent report of O-GlcNAc on Notch extracellular domain (different enzyme (eOGT) than the intracellular one (OGT))

  34. O-Glycosylation of Hyl

  35. O-Glycosylation of Hyl • Found on Collagen and Adiponectin (which has a “collagen-like” domain) • Glycosylation Essential for Basement Membrane Formation in Tissues • Modulates Collagen Cross-linking? • Other proteins with modification?

  36. The Glycosaminoglycans After Esko, J

  37. O-Glycosylation -less studied (until recently?) -tools to study are underdeveloped -in many cases, clearest functional data (not including folding/quality control)

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