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