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ESSENTIALS OF GLYCOBIOLOGY LECTURE 27 GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS May 16, 2002

ESSENTIALS OF GLYCOBIOLOGY LECTURE 27 GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS May 16, 2002 Hud Freeze. MUTANTS IN YEAST. Small Precursors Man-6-P Man-1-P Dol-P-Man Dol-P-Glc Intermediate Precursors Lipid-linked oligosaccahride (all Transferases) Oligosaccharyl transferase

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ESSENTIALS OF GLYCOBIOLOGY LECTURE 27 GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS May 16, 2002

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  1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 27 GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS May 16, 2002 Hud Freeze

  2. MUTANTS IN YEAST Small Precursors Man-6-P Man-1-P Dol-P-Man Dol-P-Glc Intermediate Precursors Lipid-linked oligosaccahride (all Transferases) Oligosaccharyl transferase Oligosaccharide Processing/ Mannan Synthesis Extension O-linked chains Glycophospholipid anchors

  3. HOW MANYGENES TO MAKE AND ADD AN ASN-LINKED CHAIN? IN YEAST? IN MAMMALIAN CELLS?

  4. N-GLYCOSYLATION PATHWAY IN YEAST G U D P UDP U D P - G D P C T P C D P G D P - M Cytoplasm G M ALG5 SEC59 DPM1 P P P P P P O-linked mannose GPI farnesyl- PP dolichol M G Lumen oligosaccharyl transferase STT3 OST1 WBP1 OST3 OST6 SWP1 OST2 OST5 OST4 G G G G G G M M G G G M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M PP M M M M M M M M ALG3 ALG9 ALG6 ALG8 ALG10 ALG12 P ALG1 ALG2 ALG7 M M = G l u c o s e = D o l i c h o l G M G D P GDP M UMP M = G l c N A c = M a n n o s e M M Modified from Burda and Aebi, 1999

  5. normal - 1 - 2 Carboxypeptidase Y (CPY) in yeast: 63 kDa vacuolar form CPY glycoform yAlg6p hAlg6p vector

  6. Do the mutations affect the function of the protein? A333V 56-86 KKXX Y131H S308R I299 F304S I299* F304S Y131H** F304S* A333V* S308R** CPY glycoform 57- 86 hALG6 vector normal - 1 - 2 * not conseved ** semi-conserved

  7. GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONS EXPRESSED IN alg6-YEAST + ALG6 – ALG6 CDG-Ic patient Y131H S308R Y131H S308R A333V CPY - 1 - 2 CPY - 1 - 2

  8. YEAST MUTANTS IN OLIGOSACCHARIDE PROCESSING

  9. PROCESSING OF N-LINKED GLYCANS IN YEAST

  10. PROCESSING OF N-LINKED GLYCANS IN YEAST

  11. O-LINKED GLYCOSYLATION IN YEAST

  12. GENERAL STRUCTURE OF GPI ANCHOR

  13. GPI ANCHOR IN YEAST--NOTE CERAMIDE SUBSITUTES FOR ONE OF THE ACYL GROUPS

  14. ONE STEP UP FOR PHOSPHORYLATION

  15. ALL DICTYOSTELIUM LYSOSOMAL GLYCOSIDASES HAVE MAN-6-P

  16. DEVELOPMENT IN DICTYOSTLIUM

  17. N-LINKED PROCESSING OCCURS DURING DEVELOPMENT

  18. O-LINKED GLYCOSYLATION--ANOTHER STEP UP Yeast - Man-Ser/Thr Dictyostelium GlcNAc~O - Not same as O-GlcNAc GlcNAc-1-P-Ser - Thr __ __

  19. GlcNAc-1-P-proteins Man-6-P-proteins Sort to different vesicles O-Linked Glycosylation - Continued Phosphoglycosylation UDP-GlcNAc+Cysteine ProteinaseGlcNAc-1-P-Cysteine Proteinase Recognition? Requires two features: 1.) acceptor site 2.) conformation of protein GlcNAc-1-P and Man-6-P modify mutually exclusive Sets of lysosomal enzymes---Why?

  20. LYSOSOMAL ENZYMES SORT TO DIFFERENT COMPARTMENTS BASED ON SUGAR CHAINS GlcNAc-1-P Man-6-P

  21. BACTERIA Man-6-P GlcNAc-1-P 3’ No chase 15’ No chase

  22. BACTERIA Man-6-P CHASE GlcNAc-1-P 15’ 30’ 60’

  23. GlcNAc-1-P and Man-6-P Remain segregated during chase 3’ pulse bacteria 3’ pulse 30’chase 95% of cells Why do they do this ???? 3’ pulse 30’chase 5% cells

  24. LABEL THE Y-AXIS AND GET A CLUE 6.5 4.5 90 15 30 60 Time (min)

  25. VARIOUS TYPES OF O-GLYCOSYLATION IN DICTY MUD166 Fuc aFuc aGlcNAc P Ser Ser GA-XIII epitope GlcNAc dependent Antibodies Show Their Power MUD62/mAb 83.5 MUD3-MUD141 mAb 83.5 aGlcNAc bFuc P P GA-XII epitope Partly Fuc depending Ser Ser GA-X epitope Fuc dependent

  26. Phosphoglycosylation

  27. Phosphoglycans in Leishmania LPG - Lipophosphoglycan - Lipid anchored to cell membrane - Composed of glycan core, repeating disaccharide (Galb1,4Mana1-P-6)n and Cap PG - Phosphoglycan-composed of cap and repeating units by biosynthetic pathway is unknown. SAP - Secreted acid phosphatase - contains SER/THR rich domain where phosphoglycosylation occurs PPG - Proteophosphoglycan - phosphoglycosylated and may be used to imobilize the parasite in the appropriate regions of the sandfly gut.

  28. Examples of Phosphoglycosylation in Leishmania I can crop the legend and make it bigger still

  29. Examples of Phosphoglycosylation in Leishmania

  30. TRANSPORTERS IN THE GOLGI

  31. SUMMARY UNICELL MODELS HAVE ADVANTAGES/DISADVANTAGES UNUSUAL STRUCTURES ARE OFTEN GOOD ANTIGENS MODELS PROVIDE EVOLUTIONARY INFORMATION FOR PATHOGENS, UNUSUAL STRUCTURES AND PATHWAYS MAY PROVIDE NOVEL APPROACHES TO DRUG DESIGN

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