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

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Presentation on theme: "ESSENTIALS OF GLYCOBIOLOGY LECTURE 13 OTHER TYPES OF GOLGI GLYCOSYLATION Hud Freeze."— Presentation transcript:

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  -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 OligosaccharidesConcentration (mg/l) Xyl  1,3Glc 5-10 *Fuc  1,2Glc 5-10 Glycopeptides Xyl  -Ser 1 Gal  -Hyl12 GlcNAc  -Asn 4 Glc  1,3Fuc  -Thr0.2 *Not reported in proteins

6 IDENTIFICATION OF UNUSUAL TYPES OF O-GLYCANS

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

8 NH 2 - -COOH = Cys = O-glucose site = O-fucose site X S X P X X XX SX X X XX X X D/N Y/F = ß-hydroxyaspartate/asparagine site EGF MODULE WITH SIGNATURE CYS RESIDUES

9 1--Glc  -Ser 2--Fuc  -Ser 3--  -hdroxy-Asp/Asn EGF Module of human Factor IX

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

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

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

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 O-fucose O-glucose both Notch retains  Fucose and  Glucose glycosylation motifs

16 EGF-like repeat with O-glucose site (C 1 XS/TXPC 2 ) EGF-like repeat with O-fucose site (C 2 X 4-5 S/TC 3 ) EGF-like repeat with both O-fucose and O-glucose sites EGF-like repeats modified by Fringe L L AA A A 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 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? Fuc  Ser/Thr+UDP-GlcNAcGlcNAc ,1,3Fuc  Ser/Thr

20 O-FucT-1

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

22 1--Glc  -Ser 2--Fuc  -Ser 3--  -hdroxy-Asp/Asn EGF Module of human Factor IX

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

24 Gal  1,4GlcNAc ,1,3Fuc  That requires  1,4GalT, the usual one for N-linked chains DON’T FORGET Glc ,1,3Fuc  Thr FOUND IN HUMAN URINE AND CHO CELLS ADDITIONAL FUNCTIONS?

25 NH 2 - -COOH S/T WWW X X XXG = Cys = O-fucose site = C-mannosylation site THROMBOSPONDIN TYPE 1 REPEATS

26 C-Mannosylation - Novel C-C bond 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 WXXW--

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

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

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

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

34 Muscular Dystrophies Walker Warburg Syndrome--POMT1 mutations cause 30% MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a  1,2GlcNAc 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  -dystroglycan glycosylation and not  -dystroglycan Hereditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

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

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

37 Muscular Dystrophies Walker Warburg Syndrome--POMT1 mutations cause 30% MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a  1,2GlcNAc 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  -dystroglycan glycosylation and not  -dystroglycan Hereditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia Dystroglycan has altered glycans--

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


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