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

3. 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

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

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

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

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

8. 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

9. YEAST MUTANTS IN OLIGOSACCHARIDE PROCESSING

10. PROCESSING OF N-LINKED GLYCANS IN YEAST

11. PROCESSING OF N-LINKED GLYCANS IN YEAST

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

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

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

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

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

25. Why do they do this ???? 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

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

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

29. Phosphoglycosylation

30. 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.

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