ESSENTIALS OF GLYCOBIOLOGY
LECTURE 27GLYCOBIOLOGY OF MODEL SINGLE CELL ORGANISMS
May 16, 2002
Hud Freeze
MUTANTS IN YEAST
Small Precursors Man-6-PMan-1-PDol-P-ManDol-P-Glc
Intermediate Precursors Lipid-linked oligosaccahride (all Transferases)Oligosaccharyl transferase
Oligosaccharide Processing/ Mannan Synthesis Extension O-linked chains
Glycophospholipid anchors
HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?HOW MANY GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?GENES TO MAKE AND ADD AN ASN-LINKED CHAIN?
IN YEAST? IN MAMMALIAN CELLS?
oligosaccharyltransferase
STT3OST1WBP1OST3OST6SWP1
OST2OST5OST4
MM
MMM
dolichol
CTPCDP
SEC59
P
Lumen
P
M
MMMM
M
MMMM
M
GGG
MMM
M M
MMM
MM
MM
MM
MMM
ALG3 ALG9
MM
MM
ALG6 ALG8 ALG10
DPM1MP
G GDPDP-M
MP
UDP- UDP
ALG5P
GP
GP
O-linked mannoseGPI
M
G
MM
MMM
MG
MM
M
MM
MM
MMM
G
= GlcNAc
= Dolichol
M = Mannose
= Glucose
Cytoplasm
farnesyl-PP
ALG7
UMP
UDP GDPPGDM
ALG1ALG2
MMM
MMM
MMM
MMM
GM
MMMMGGG
M
MMM
M
ALG12
Modified fromBurda and Aebi, 1999
PP
G
N-GLYCOSYLATION PATHWAY IN YEASTN-GLYCOSYLATION PATHWAY IN YEAST
63 kDa vacuolar form
Carboxypeptidase Y (CPY) in yeast:Carboxypeptidase Y (CPY) in yeast:
CPY glycoform
normal- 1- 2
yAlg6p hAlg6p vector
Do the mutations affect the function of the protein?Do the mutations affect the function of the protein?
S308RY131H F304S
KKXX
A333V
I299
hALG6 Y131H** vectorCPY
glycoformnormal- 1- 2
I299*F304S F304S* A333V* S308R**
57-86
* not conseved ** semi-conserved
56-86
GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONSEXPRESSED IN alg6- YEAST
GROWTH AND STRESS AFFECT IMPACT OF hALG6 MUTATIONSEXPRESSED IN alg6- YEAST
CPY
+ALG6
A333VY131HS308R
CDG-Ic patient
Y131H
–ALG6
- 1- 2
S308R
CPY
- 1- 2
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 SUBSITUTESFOR ONE OF THE ACYL GROUPS
ONE STEP UP FOR PHOSPHORYLATION
ALL DICTYOSTELIUM
LYSOSOMAL GLYCOSIDASES HAVE MAN-6-P
DEVELOPMENT INDICTYOSTLIUM
N-LINKED PROCESSING OCCURS DURING DEVELOPMENT
O-LINKED GLYCOSYLATION--ANOTHER STEP UP
Yeast - Man-Ser/Thr
Dictyostelium
GlcNAc~O - Not same as O-GlcNAc
GlcNAc-1-P-Ser
-
Thr
____
O-Linked Glycosylation - Continued
Phosphoglycosylation
UDP-GlcNAc+Cysteine ProteinaseGlcNAc-1-P-Cysteine ProteinaseRecognition?
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 exclusiveSets of lysosomal enzymes---Why?
GlcNAc-1-P
Man-6-P
LYSOSOMAL ENZYMES SORT TO DIFFERENTCOMPARTMENTS BASED ON SUGAR CHAINS
3’
15’
BACTERIAGlcNAc-1-P Man-6-P
No chase
No chase
GlcNAc-1-P Man-6-PBACTERIA
15’
30’
60’
CHASE
GlcNAc-1-P and Man-6-PRemain segregated duringchase
3’ pulsebacteria
3’ pulse30’chase95% of cells
3’ pulse30’chase5% cells
Why do they do this????
6.5
4.5
Time (min)15 30 60 90
LABEL THE Y-AXIS AND GET A CLUE
VARIOUS TYPES OF O-GLYCOSYLATION IN DICTY
Antibodies Show Their Power
Fuc
SerP
GlcNAc
Ser
MUD166
GA-XIII epitopeGlcNAc dependent
GA-XII epitopePartly Fuc depending
P
Fuc
Ser
GlcNAc
P
Fuc
Ser
GA-X epitopeFuc dependent
mAb 83.5
MUD62/mAb 83.5
MUD3-MUD141
Phosphoglycosylation
Phosphoglycans in Leishmania
LPG - Lipophosphoglycan- Lipid anchored to cell membrane- Composed of glycan core, repeating disaccharide
(Gal1,4Man1-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
SUMMARYUNICELL MODELS HAVE ADVANTAGES/DISADVANTAGES
UNUSUAL STRUCTURES ARE OFTEN GOOD ANTIGENS
MODELS PROVIDE EVOLUTIONARY INFORMATION
FOR PATHOGENS, UNUSUAL STRUCTURES AND PATHWAYSMAY PROVIDE NOVEL APPROACHES TO DRUG DESIGN
