Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "ESSENTIALS OF GLYCOBIOLOGY LECTURE 27 GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS May 16, 2002 Hud Freeze."— Presentation transcript:

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

2

3 MUTANTS IN YEAST Small PrecursorsMan-6-P Man-1-P Dol-P-Man Dol-P-Glc Intermediate PrecursorsLipid-linked oligosaccahride (all Transferases) Oligosaccharyl transferase Oligosaccharide Processing/Mannan Synthesis ExtensionO-linked chains Glycophospholipid anchors

4 GENES TO MAKE AND ADD AN ASN-LINKED CHAIN? HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN? IN YEAST? IN MAMMALIAN CELLS?

5 oligosaccharyl transferase STT3 OST1 WBP1 OST3 OST6 SWP1 OST2 OST5 OST4 M M M M M dolichol CTPCDP SEC59 P Lumen P M M M M M M M M M M M G G G M M M MM M MM M M M M M M M MM ALG3ALG9 M M M M ALG6ALG8ALG10 DPM1 M P G GDP DP- M M P UDP- UDP ALG5 P G P G P O-linked mannose GPI M G M M M MM M G M M M M M M M M MM G = GlcNAc =Dolichol M =Mannose = Glucose Cytoplasm farnesyl- PP ALG7 UM P UDP GDP PGD M ALG1 ALG2 M M M M M M M M M M M M G M M M M M G G G M M MM M ALG12 Modified from Burda and Aebi, 1999 PP G N-GLYCOSYLATION PATHWAY IN YEAST

6 63 kDa vacuolar form Carboxypeptidase Y (CPY) in yeast: Carboxypeptidase Y (CPY) in yeast: CPY glycoform normal yAlg6phAlg6pvector

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

8 GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONS EXPRESSED IN alg6 - YEAST GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONS EXPRESSED IN alg6 - YEAST CPY + ALG6 A333V Y131H S308R CDG-Ic patient Y131H – ALG S308R CPY

9 YEAST MUTANTS IN OLIGOSACCHARIDE PROCESSING

10 PROCESSING OF N-LINKED GLYCANS IN YEAST

11

12 O-LINKED GLYCOSYLATION IN YEAST

13 GENERAL STRUCTURE OF GPI ANCHOR

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

15

16 ONE STEP UP FOR PHOSPHORYLATION

17 ALL DICTYOSTELIUM LYSOSOMAL GLYCOSIDASES HAVE MAN-6-P

18 DEVELOPMENT IN DICTYOSTLIUM

19 N-LINKED PROCESSING OCCURS DURING DEVELOPMENT

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

21 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-proteins Man-6-P-proteins Sort to different vesicles GlcNAc-1-P and Man-6-P modify mutually exclusive Sets of lysosomal enzymes---Why?

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

23 3’3’ 15’ BACTERIA GlcNAc-1-P Man-6-P No chase

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

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

26 Time (min) LABEL THE Y-AXIS AND GET A CLUE

27 VARIOUS TYPES OF O-GLYCOSYLATION IN DICTY Antibodies Show Their Power Fuc Ser P  GlcNAc Ser MUD166 GA-XIII epitope GlcNAc dependent GA-XII epitope Partly Fuc depending P  Fuc Ser  GlcNAc P  Fuc Ser GA-X epitope Fuc dependent mAb 83.5 MUD62/mAb 83.5 MUD3-MUD141

28

29 Phosphoglycosylation

30 Phosphoglycans in Leishmania LPG-Lipophosphoglycan -Lipid anchored to cell membrane -Composed of glycan core, repeating disaccharide (Gal  1,4Man  1-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.

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

32 Examples of Phosphoglycosylation in Leishmania

33 TRANSPORTERS IN THE GOLGI

34 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

35


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

Similar presentations


Ads by Google