o-glycans. what is an o-glycan? ser/thr termed an “o-glycan”
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O-Glycans
What is an O-Glycan?
Ser/Thr Ser/Thr Ser/ThrSer/Thr
termed an “O-Glycan”
Mucins are heavily O-glycosylated
Mucin Production in the Intestinal Crypts
UDP-
Ser/Thr
ppGalNAcT-2
ppGalNAcT-1
ppGalNAcT-3
ppGalNAcT-11
?
Ser/Thr
Multiple polypeptide GalNAc transferases InitiateProtein O-glycosylation
Core 1 and Core 2 O-Glycan Synthesis
Core 3 and Core 4 O-Glycan Synthesis
3
Ser/Thr
Ser/Thr6
Ser/Thr
Core 5GalNAcT
Core 6GlcNAcT
Core 7GalNAcT 6
Ser/Thr
Unusual Core O-Glycan Structures Reported
What happens to O-glycan formation and mice that lack Core 2 GlcNAcT?
Core 2 GlcNAcT Activity Persists in Various Mouse Tissues
0
40
60
80
100
120
20
0
1
2
3
4
cells/mlx103
Core 2 GlcNAcT Deficiency Results in Leukocytosis
WBC
cells/mlx103
Neu
Lym EosMon
wt/∆
wt
∆/∆
Loss of Selectin Ligands in Core 2 GlcNAcT Null Mice
50
100
150
200
250
300
350
400
500
450
Time (hrs.) Post-Thioglycollate Stimulus
Impaired Neutrophil Recruitment to the Peritoneum ofMice Lacking Core 2 GlcNAcT
550
600
wt
null
Neut. #x104
0 42
-/-
wt/wt
-/-
wt/wt
∆/∆
wt/wt
-/-
wt/wt
-/-
wt/wt
wt/wt -/-
Neutrophil Recruitment in a model of peritonitis
100
%WT
Defic. P-Sel L-Sel E-Sel P/E FT7 C2-1
0
40
0
2
4
6
8
10
12
14
∆/∆wt/wt
Normal Leukocyte Abundance and Homing in Lymph Nodesof Mice Lacking the Core 2 GlcNAcT Gene
0
1.5
1.0
0.5
∆/∆
wt/wt
PLNMLN PP
Cellsx106
wt null
MN PN PP
CMFDA+cells
Core 2 O-Glycans Participate in L-Selectin Ligand Formationin Lymph Node High Endothelial Venules (HEVs)
L-selectin
Meca-79Meca-79
L-selectin
WT Null
Core 1 O-Glycan Extension by a Novel GlcNAcT EnzymeFirst Observed in the Core 2 GlcNAcT Deficient Mouse
Ser/Thr
3
3
Core 1 extensionGlcNAcT
Selectin ligand
At Least Three Different Core 2 GlcNAcT Enzymes C2 GlcNAcT-I, -II, and -III
Initiate Core 2 O-Glycan Formation
Core 1 Extension Products are Found in the Absence of Core 2 GlcNAcT-I
Core 2 GlcNAcT-I is Essential for Myeloid Homeostasisand the inflammation Response Involving Neutrophils
Regulation of Core 2 O-Glycan Formation
Competition between Golgi Glycosyltransferases:
The ST3Gal-I Null Mouse
Hematoxylin PNA
Thymic Cortical and Medullary Compartments Delineated byHistochemical Staining and PNA Lectin Binding
Cortex
Medulla
ST3Gal-I Sialyltransferase and Core 1 O-Glycan Structurein T Cell Development and Activation
Ser/Thr
PNA
Ser/Thr
ST3Gal-I
THYMUS
cortex medulla
?
Ser/ThrSer/Thr
PERIPHERY
activatednaive
PNA
100X
200X
WT ST3Gal-I Null
PNA Binding (brown) in Thymus
Thymic CD8-MHC Binding Modulated by ST3Gal-I
Cortex
MedullaSS
SS
CD8CD8
MHC I 3protrudingloop
Thymic CD8-MHC Binding Modulated by ST3Gal-I
Cortex
MedullaS S
SS
CD8CD8
MHC I 3protrudingloop
Cell #% of WT
100
thymus spleen
50
blood lymphnodes
25
CD4 CD8
75
Deficiency of Peripheral CD8+ T Cells in ST3Gal-I Null Mice
0
20
40
100
60
80
% Annexin V+
Time (hrs)
-0 24
1B110 24 240
Immune Activation Blocks CD8+ T Cell Apoptosis in ST3Gal-I Null Mice
anti-CD3pretreatment
wt
∆/∆ 1B11
T Cell O-Glycans in the Absence of ST3Gal-I
Ser/Thr
43
3 6
spleen
wtwt/∆∆/∆
pm
ol /
mg
, hr
Core 2 GlcNAcTActivity
thymus
6
4
2
0
3
Ser/Thr
Core 1GaT
ST3Gal-I Expression Suppresses Core 2 O-Glycan Formationin the T Cell Golgi Apparatus
ST3Gal-I
ST6GalNAcT36
6Core 2GlcNAcT-I
X
4
3
Ser/Thr
Ser/ThrSer/Thr
Ser/Thr
75
50
25
00 6 12 24 48 72
% PNAhigh
Hours post-TCR Activation
100
Core 1 and Core 2 O-Glycans in CD8+ T Cell Activation
% 1B11high
naive
ST3Gal-I in Peripheral CD8+ T Lymphocyte Homeostasis: Role in Post-Immune Apoptosis
immunestimulus
ST3Gal-I: ON
activated
ST3Gal-I: OFF
viablememory
ST3Gal-I: ON
apoptosis
stimulusdepleted
ST3Gal-I: OFF
>90%
immunestimulusST3Gal-I
deficiency
‘activated’ O-glycan structure
apoptoticmemory
ST3Gal-I in Peripheral CD8+ T Lymphocyte Homeostasis: Role in Post-Immune Apoptosis
E N Z Y M E
polypeptide GalNAcT-1
polypeptide GalNAcT-8
Core 2 GlcNAcT
1-4 GalT
FucT-IV
FucT -VII
1-3 GalT (Gal3T)
ST3Gal-I
ST3Gal-II
ST3Gal-III
ST3Gal-IV
P H E N O T Y P E / D ISE A SE
B lymphocyte deficiency in nodes
viable/under study
m yeloid leukocytosis and defect in inflammation
m ultiple defects including epithelial and endocrine
abnormalities with frequent post-natal lethality
partial inflammation response deficit, collaboration with FucT-IV
general leukocytosis , lymphoid homing defect , and
inflammatory response deficit
cataracts
cytotoxic T cell deficiency by apoptosis
viable/under study
inflammation deficit, vW F and platelet deficiency
Induced O-glycan Deficiencies in the Mouseand Biological Effects
not O-glycan specific
*
*
*
****
*
*behavioral defects, under study
Branch Specificity in O-Glycan Structure-Function Relationships
Core 1 O-GlycansST3GalT-I-dependent
Core 2 O-Glycans
CD8+ T cell homeostasis
Myeloid cell homeostasisand inflammation response
3 6
Ser/Thr
N- and O-Glycans Modulate Physiologic Systems
Glycosyltransferase and Glycosidase Specificity:
Regulated Expression
Competition with other Golgi Enzymes
Different Substrate (protein?) Specificities
Multiple isozymes