Transcript Lecture_27.pps

ESSENTIALS OF GLYCOBIOLOGY
LECTURE 27
GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS
May 16, 2002
Hud Freeze
System
Glycobiology Model Systems
Advantages
Disadvantages
Yeast
Genome sequenced,
Fast generation time
Many Mutants
Conserved Pathways
Little N-linked processing
Mannose-based O-linked
No Glycosphingolipids (GSL)
No GAGS or Proteoglycans
No Development or Behavior
Dictyostelium
Growth and multicellular development No GAGS
Fast development
Antigenic oligo chains
No Glycosphingolipids
Some behavior
Unusual O-linked
Genome Sequenced
Few structures
C. elegans
Multicellular development
Cell Fates
Extensive N-linked oligo processing
Beginning of O-GalNAc linked
Beginning of Glycosphingolipids
Genome sequenced
Limited Structural Information
Drosophilia
Excellent genetic system
Neurological Development-assays
Proteoglycan (Heparan Sulfate)
Genome Sequenced
No complex sialylated N-linked glycans
GSL are unusual
Few structures analyzed
Xenopus
Good for early development
Fertilization, protein expression
Long generation time
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/
Extension
Mannan Synthesis
O-linked chains
Glycophospholipid anchors
HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?
IN YEAST? IN MAMMALIAN CELLS?
N-GLYCOSYLATION PATHWAY IN YEAST
Cytoplasm
SEC59
farnesylPP
UDP-G
CTP CDP G DP- M GDP
P
DPM1
G
ALG5
P
P
M
P
P
M
dolichol
O-linked mannose
GPI
P
G
oligosaccharyl
transferase G
Lumen
M
M
M
M
M
M
M M
M M
M
ALG3
P
ALG7
UDP
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
ALG9
ALG12
ALG6
G
G
M
M
M
ALG8
G
STT3
G
OST1
G
WBP1
M M M M M OST3
M M M M M OST6
M M SWP1
M
M
M
OST2
ALG10
G
G
M M M
M M M
M M
M
PP
OST5
OST4
ALG1
ALG2
M
UDP UM GDP GDP M
P M
M
M
M
= Dolichol
G = Glucose
= GlcNAc
M = Mannose
Modified from
Burda and Aebi, 1999
Carboxypeptidase Y (CPY) in yeast:
63 kDa vacuolar form
yAlg6p
hAlg6p
vector
CPY
glycoform
normal
-1
-2
Do the mutations affect the function of the protein?
56-86
A333V
KKXX
Y131H
hALG6
I299*
F304S
F304S*
I299 F304S S308R
Y131H**
A333V*
S308R**
*
5786
not conseved
vector
CPY
glycoform
normal
-1
-2
** semi-conserved
GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONS
EXPRESSED IN alg6- YEAST
+
ALG6
CDG-Ic patient
Y131H
A333V S308R Y131H S308R
CPY
-1
-2
CPY
-1
-2
–
ALG6
YEAST MUTANTS IN OLIGOSACCHARIDE PROCESSING
PROCESSING OF N-LINKED GLYCANS IN YEAST
PROCESSING OF N-LINKED GLYCANS IN YEAST
O-LINKED GLYCOSYLATION IN YEAST
GENERAL STRUCTURE OF GPI ANCHOR
GPI ANCHOR IN YEAST--NOTE CERAMIDE SUBSITUTES
FOR ONE OF THE ACYL GROUPS
ONE STEP UP FOR PHOSPHORYLATION
ALL
DICTYOSTELIUM
LYSOSOMAL
GLYCOSIDASES
HAVE MAN-6-P
DEVELOPMENT IN
DICTYOSTLIUM
N-LINKED PROCESSING OCCURS DURING DEVELOPMENT
O-LINKED GLYCOSYLATION--ANOTHER STEP UP
Yeast - Man-Ser/Thr
Dictyostelium
GlcNAc~O - Not same as O-GlcNAc
Thr
GlcNAc-1-P-Ser
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
LYSOSOMAL ENZYMES SORT TO DIFFERENT
COMPARTMENTS BASED ON SUGAR CHAINS
GlcNAc-1-P
Man-6-P
BACTERIA
GlcNAc-1-P
Man-6-P
3’
No chase
15’
No chase
BACTERIA
GlcNAc-1-P
Man-6-P
CHASE
15’
30’
60’
3’ pulse
bacteria
GlcNAc-1-P and Man-6-P
Remain segregated during
chase
3’ pulse
30’chase
95% of cells
Why do they do this
????
3’ pulse
30’chase
5% cells
LABEL THE Y-AXIS AND GET A CLUE
6.5
4.5
15
30
60
90
Time (min)
VARIOUS TYPES OF O-GLYCOSYLATION IN DICTY
Antibodies Show Their Power
MUD62/mAb 83.5
MUD3-MUD141
mAb 83.5
aFuc
bFuc
aGlcNAc
MUD166
P
P
P
Ser
Ser
Ser
GA-X epitope
Fuc dependent
Fuc
aGlcNAc
Ser
GA-XII epitope
Partly Fuc depending
GA-XIII epitope
GlcNAc dependent
Phosphoglycosylation
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.
Examples of Phosphoglycosylation in Leishmania
I can crop the legend and make it bigger still
Examples of Phosphoglycosylation in Leishmania
TRANSPORTERS IN THE GOLGI
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