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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-linked Mannose-based glycans are abundant in brain

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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-linked Mannose-based glycans are abundant in brain • These chains are prominent on a-dystroglycan • Altered O-Mannose glycans cause Muscular Dystrophy • O-Fucose, O-Glucose found in EGF domains • Notch signaling pathways depend on glycosylation • 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 • Primitive eukaryotic function - selection and adaptation to specific needs in mammals

  5. Urinary Oligosaccharides and Glycosides Oligosaccharides 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. 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

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

  8. 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

  9. 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 • Could these be involved in Signaling????

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

  11. 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…....

  12. 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.

  13. Notch and its ligands both contain EGF domains Delta Serrate Notch FRINGE GENES SIGNALS SIGNALS SIGNALS SIGNALS SIGNALS SIGNALS SIGNALS

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

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

  16. Galb1,4GlcNAcb,1,3Fuca MINIMAL GLYCAN INVOLVED IN NOTCH SIGNALING 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?

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

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

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

  20. Muscular Dystrophies *MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a b1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Encodes a glycosylt’ase--O-mannosyl transferase(?) Fukutin Related protein--(19q13.3) dystroglycan misglycosyaltion and laminin deficiency Overexpression of a specific b1,4GalNAc transferase reverses MD in a model of Duchenne MD (dystrophin) Common Features: Affects a-dystroglycan glycosylation and not b-dystroglycan Heriditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

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

  22. Muscular Dystrophies MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a b1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Encodes a glycosylt’ase--O-mannosyl transferase(?) *Fukutin Related protein--(19q13.3) dystroglycan misglycosyaltion and laminin deficiency Overexpression of a specific b1,4GalNAc transferase reverses MD in a model of Duchenne MD (dystrophin) Common Features: Affects a-dystroglycan glycosylation and not b-dystroglycan Heriditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

  23. Dystroglycan in Myd Mouse

  24. Muscular Dystrophies MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a b1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Encodes a glycosylt’ase--O-mannosyl transferase(?) Fukutin Related protein--(19q13.3) dystroglycan misglycosyaltion and laminin deficiency *Overexpression of a specific b1,4GalNAc transferase reverses MD in a model of Duchenne MD (dystrophin) Common Features: Affects a-dystroglycan glycosylation and not b-dystroglycan Heriditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

  25. O-Mannose: An Emerging Family of Glycans Over expressed

  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

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

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