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ESSENTIALS OF GLYCOBIOLOGY LECTURE 13 OTHER TYPES OF GOLGI GLYCOSYLATION Hud Freeze

ESSENTIALS OF GLYCOBIOLOGY LECTURE 13 OTHER TYPES OF GOLGI GLYCOSYLATION Hud Freeze. SUMMARY AND TAKE HOME MESSAGES. “Rare” forms of glycosylation are not rare or insignificant Technical limitations slowed their identification O-Fucose, O-Glucose found in EGF domains

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ESSENTIALS OF GLYCOBIOLOGY LECTURE 13 OTHER TYPES OF GOLGI GLYCOSYLATION Hud Freeze

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  1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 13 OTHER TYPES OF GOLGI GLYCOSYLATION Hud Freeze

  2. SUMMARY AND TAKE HOME MESSAGES • “Rare” forms of glycosylation are not rare or insignificant • Technical limitations slowed their identification • O-Fucose, O-Glucose found in EGF domains • Notch signaling pathways depend on glycosylation • Pattern recognition may combine peptide+glycans • O-Mannose glycans • Are abundant in brain and muscle • Prominent on a-dystroglycan • Altering O-mannose glycans causes Muscular Dystrophy • Other forms of glycosylation are likely to have functions

  3. RARE FORMS OF GLYCOSYLATION • Discovery • Minor component in an abundant source • Thorough analysis of a well known or important molecules • Antibody against a “glycan” • Dedicated analysis of mixtures of glycans, proteins or organs • Chance

  4. RARE FORMS OF GLYCOSYLATION • Roadblocks to Discovery • Degradation and analysis is difficult • Minor amounts • Biosynthetic route is unknown • Tools required are often state of the art level • Significance • Ranges from unknown-----> critically important

  5. Urinary Oligosaccharides and Glycosides Oligosaccharides Concentration (mg/l) Xyla1,3Glc 5-10 *Fuca1,2Glc 5-10 Glycopeptides Xylb-Ser 1 Galb-Hyl 12 GlcNAcb-Asn 4 Glcb1,3Fuca-Thr 0.2 *Not reported in proteins

  6. IDENTIFICATION OF UNUSUAL TYPES OF O-GLYCANS

  7. C-X-S-X-P-C C-X-X-G-G-T/S-C C-X-D/N-X-X-X-Y/F-C-X-C EGF MODULE AND ASSOCIATES

  8. EGF MODULE WITH SIGNATURE CYS RESIDUES S X NH2- X P 1 2 X X 3 X S X X D/N X X X 5 X 4 X X Y/F = Cys = O-glucose site 6 -COOH = O-fucose site = ß-hydroxyaspartate/asparagine site

  9. EGF Module of human Factor IX 1--Glcb-Ser 2--Fuca-Ser 3--b-hdroxy-Asp/Asn

  10. What are the functions of these modifications? Little effect on half-life or activity of clotting proteins How about other proteins with EGF modules? Now it gets interesting

  11. FRINGE GENES SIGNALS SIGNALS SIGNALS SIGNALS Notch and its ligands both contain EGF domains Delta Serrate (Jagged) Notch SIGNALS SIGNALS SIGNALS

  12. CSL Nucleus NOTCH LIGAND BINDING AND SIGNALING Sending Cell Ligand Notch TACE g-secretase Receiving Cell

  13. Fringe proteins modify Notch/ligand binding and signaling Jagged Jagged Jagged Notch + lunatic + manic Notch Notch Signals

  14. Notch - an EGF Signaling Protein • Signaling molecule from C. elegans  Humans • Signals induced in binding to Delta or Jagged (serrate) on adjacent cells • Signaling defects in Notch cause abnormal development, leukemia and a complex disease of Cerebral Artiopathy and Infarcts • Human Notch-1 (of 4) contains 12 O-Fucose and 17 O-glucose modification consensus sites

  15. Notch retains aFucose and bGlucose glycosylation motifs O-fucose O-glucose both

  16. 1 5 10 15 20 25 30 35 EGF-like repeat with O-glucose site (C1XS/TXPC2) EGF-like repeat with O-fucose site (C2X4-5S/TC3) EGF-like repeat with both O-fucose and O-glucose sites A A A A L L EGF-like repeats modified by Fringe Lin12/Notch repeat

  17. Are the Sugar Chains Important for Anything? • Half of the glycosylation sites are conserved across species (3, 4, 10, 12-14, 13, and 14 all contain conserved sites). • The EGF modules 11 and 12 are essential for binding to Delta. Modules 10, 12, 13, and 14 all contain conserved sites • Abnormal wing vein mutations occur in modules 24-29. • Human disorders have been tracked to mutations in Notch 3. • Fringe proteins differentially modify Notch binding to Delta and Serrate. Wonder what fringe is…....

  18. Are the Sugar Chains Important for Anything? • Mammalian fringe homologs exist--manic, lunatic, radical • Fringe must be expressed in the same cell as Notch to exert its effect. Fringe is secreted.

  19. FRINGE IS A GLYCOSYL TRANSFERASE!!! ARE YOU REALLY SURPRISED? Fuca-Ser/Thr+UDP-GlcNAc GlcNAcb,1,3Fuca-Ser/Thr

  20. O-FucT-1

  21. Fringe proteins modify Notch/ligand binding and signaling Lunatic and manic modify different EGF modules Jagged Jagged Jagged Notch +lunatic + manic Notch Notch Signals

  22. EGF Module of human Factor IX 1--Glcb-Ser 2--Fuca-Ser 3--b-hdroxy-Asp/Asn

  23. BAH--b ASP/ASN HYDROXYLATION Mice have developmental Abnormalities and are also prone to intestinal polyps

  24. Galb1,4GlcNAcb,1,3Fuca That requires b1,4GalT, the usual one for N-linked chains DON’T FORGET Glcb,1,3Fuca-Thr FOUND IN HUMAN URINE AND CHO CELLS ADDITIONAL FUNCTIONS?

  25. THROMBOSPONDIN TYPE 1 REPEATS NH2- W W W 1 X X S/T 3 G X X 2 -COOH 6 5 4 = Cys = C-mannosylation site = O-fucose site

  26. C-Mannosylation - Novel C-C bond WXXW-- Consensus sequence in 100’s of proteins and wide-spread in mammalian cells. Mannosyl transferase present in many cells. Antibody and structural determination are keys to finding this unusual form of protein modification

  27. Thrombospondin has Man(C)--Trp And Glc-Fuc-O-disaccharide in EGF domains Man(C)--Trp Glc-Fuc--O-Ser/Thr 368 377 420 432 423 --- 480 489 Residues implicated in binding to GAG chains Cell surface binding of Thromospondin is necessary for its anti-angiogenic function

  28. O O O - - - SO3 SO3 SO3 GLYCOSYLATION + AMINO ACID MODIFICATION A RECURRING THEME IN RECOGNITION? 42 46 57 48 51 Q - A - T - E - Y - E - Y - L - D - Y - D - F - L - P - E - T - E - P - P - 1 6 3 4 3 3 3 P-Selectin C-Type Lectin Domain Tyr-SO4 EGF-Domain Sugar chain CR-Repeat

  29. O-Mannose: An Emerging Family of Glycans About 1/3 of brain O-linked chains are O-mannose based

  30. O-Man and O-GalNAc chains N-linked chains

  31. Dystroglycan is misglycosylated in the Muscular dystrophic Myd Mouse Protein is fine Anti-Glycan Tissue-specific glycosylation

  32. Muscular Dystrophies Walker Warburg Syndrome--POMT1 mutations cause 30% MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a b1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Putative transferase in golgi (?) Fukutin Related protein--(19q13.3) Putative transferase (?) LARGE- cause of myd mouse, tandem glycosyltransfrases(?) Common Features: Affects a-dystroglycan glycosylation and not b-dystroglycan Hereditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

  33. O-Mannose: An Emerging Family of Glycans WWS-Mutated transferase

  34. O-Mannose: An Emerging Family of Glycans MEB- Mutated transferase

  35. Muscular Dystrophies Walker Warburg Syndrome--POMT1 mutations cause 30% MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a b1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Putative transferase in golgi (?) Fukutin Related protein--(19q13.3) Putative transferase (?) dystroglycan misglycosylation LARGE- cause of myd mouse, tandem glycosyltransfrases(?) Common Features: Affects a-dystroglycan glycosylation and not b-dystroglycan Hereditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia Dystroglycan has altered glycans--

  36. VARIABLE TYPES OR AMOUNTS OF GLYCOSYLATION CAN STRONGLY AFFECT BIOLOGICAL READOUT AND PHYSIOLOGY-- WATCH OUT FOR THE NEWCOMERS

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