O-Glycans

1. O-Glycans

2. What is an O-Glycan? Ser/Thr Ser/Thr Ser/Thr Ser/Thr termed an “O-Glycan”

3. Mucins are heavily O-glycosylated

4. Mucin Production in the Intestinal Crypts

5. Multiple polypeptide GalNAc transferases Initiate Protein O-glycosylation ? ppGalNAcT-11 ppGalNAcT-3 ppGalNAcT-2 ppGalNAcT-1 UDP- Ser/Thr Ser/Thr

6. Core 1 and Core 2 O-Glycan Synthesis

8. Core 3 and Core 4 O-Glycan Synthesis

9. Unusual Core O-Glycan Structures Reported 3 a Core 5 GalNAcT Ser/Thr Core 6 6 b GlcNAcT Ser/Thr Ser/Thr Core 7 GalNAcT 6 a Ser/Thr

10. What happens to O-glycan formation and mice that lack Core 2 GlcNAcT?

11. Core 2 GlcNAcT Activity Persists in Various Mouse Tissues

12. Core 2 GlcNAcT Deficiency Results in Leukocytosis 1 2 0 4 1 0 0 cells/ml x103 cells/ml x103 3 8 0 6 0 2 4 0 1 2 0 0 0 Lym WBC Mon Eos Neu wt wt/∆ ∆/∆

13. Loss of Selectin Ligands in Core 2 GlcNAcT Null Mice

14. Impaired Neutrophil Recruitment to the Peritoneum of Mice Lacking Core 2 GlcNAcT 6 0 0 5 5 0 wt 5 0 0 4 5 0 null 4 0 0 3 5 0 Neut. # x104 3 0 0 2 5 0 2 0 0 1 5 0 1 0 0 5 0 0 2 4 Time (hrs.) Post-Thioglycollate Stimulus

15. Neutrophil Recruitment in a model of peritonitis Defic. P-Sel L-Sel E-Sel P/E FT7 C2-1 w t / w t - / - w t / w t w t / w t w t / w t w t / w t w t / w t - / - - / - ∆ / ∆ - / - - / - 100 %WT 40 0

16. Normal Leukocyte Abundance and Homing in Lymph Nodes of Mice Lacking the Core 2 GlcNAcT Gene 1 4 w t / w t 1 2 ∆ / ∆ Cells x106 1 0 1 . 5 w t / w t 8 ∆ / ∆ 6 4 1 . 0 CMFDA+ cells 2 0 MN PN PP 0 . 5 wt 0 null M L N P L N P P

17. Core 2 O-Glycans Participate in L-Selectin Ligand Formation in Lymph Node High Endothelial Venules (HEVs) WT Null L-selectin L-selectin Meca-79 Meca-79

18. Core 1 O-Glycan Extension by a Novel GlcNAcT Enzyme First Observed in the Core 2 GlcNAcT Deficient Mouse Selectin ligand Core 1 extension GlcNAcT 3  3 b Ser/Thr

19. 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 Homeostasis and the inflammation Response Involving Neutrophils

20. Regulation of Core 2 O-Glycan Formation Competition between Golgi Glycosyltransferases: The ST3Gal-I Null Mouse

21. PNA Thymic Cortical and Medullary Compartments Delineated by Histochemical Staining and PNA Lectin Binding Hematoxylin Cortex Medulla

22. ST3Gal-I Sialyltransferase and Core 1 O-Glycan Structure in T Cell Development and Activation PERIPHERY THYMUS cortex medulla naive activated PNA PNA ? ST3Gal-I Ser/Thr Ser/Thr Ser/Thr Ser/Thr

23. PNA Binding (brown) in Thymus WT ST3Gal-I Null 100X 200X

24. Thymic CD8-MHC Binding Modulated by ST3Gal-I MHC I 3 a protruding loop CD8 a Cortex CD8 b S S Medulla S S

25. Thymic CD8-MHC Binding Modulated by ST3Gal-I MHC I 3 a protruding loop CD8 a Cortex CD8 b S S Medulla S S

26. Deficiency of Peripheral CD8+ T Cells in ST3Gal-I Null Mice CD4 CD8 100 75 Cell # % of WT 50 25 thymus blood lymph nodes spleen

27. Immune Activation Blocks CD8+ T Cell Apoptosis in ST3Gal-I Null Mice anti-CD3 pretreatment 100 80 60 % Annexin V+ 40 20 wt 0 0 24 0 24 0 24 ∆/∆ - 1B11 1B11 Time (hrs)

28. T Cell O-Glycans in the Absence of ST3Gal-I Core 2 GlcNAcT Activity 6 wt 3 a 4 wt/∆ pmol / mg, hr 4 b ∆/∆ 2 6 3 b b 0 Ser/Thr thymus spleen

29. X  3 b 4 Core 2 GlcNAcT-I 6 b ST3Gal-I Expression Suppresses Core 2 O-Glycan Formation in the T Cell Golgi Apparatus  3 ST6GalNAcT  6 Core 1 GaT ST3Gal-I Ser/Thr Ser/Thr b 3 Ser/Thr Ser/Thr Ser/Thr

30. Core 1 and Core 2 O-Glycans in CD8+ T Cell Activation 100 high 75 % PNA high 50 % 1B11 25 0 0 6 12 24 48 72 Hours post-TCR Activation

31. ST3Gal-I in Peripheral CD8+ T Lymphocyte Homeostasis: Role in Post-Immune Apoptosis ST3Gal-I: ON viable memory apoptotic memory naive activated stimulus depleted immune stimulus apoptosis >90% ST3Gal-I: ON ST3Gal-I: OFF ST3Gal-I: OFF immune stimulus ST3Gal-I deficiency ‘activated’ O-glycan structure

32. ST3Gal-I in Peripheral CD8+ T Lymphocyte Homeostasis: Role in Post-Immune Apoptosis

33. E N Z Y M E P H E N O T Y P E / D I S E A S E B lymphocyte deficiency in nodes p o l y p e p t i d e G a l N A c T - 1 p o l y p e p t i d e G a l N A c T - 8 v i a b l e / u n d e r s t u d y C o r e 2 G l c N A c T m y e l o i d l e u k o c y t o s i s a n d d e f e c t i n i n f l a m m a t i o n 1 - 4 G a l T b m u l t i p l e d e f e c t s i n c l u d i n g e p i t h e l i a l a n d e n d o c r i n e a b n o r m a l i t i e s w i t h f r e q u e n t p o s t - n a t a l l e t h a l i t y F u c T - I V p a r t i a l i n f l a m m a t i o n r e s p o n s e d e f i c i t , c o l l a b o r a t i o n w i t h F u c T - I V F u c T - V I I g e n e r a l l e u k 1 - 3 G a l T ( G a l 3 T ) a S T 3 G a l - I S T 3 G a l - I I S T 3 G a l - I I I S T 3 G a l - I V Induced O-glycan Deficiencies in the Mouse and Biological Effects * * o c y t o s i s l y m p h o i d h o m i n g d e f e c t , , a n d * i n f l a m m a t o r y r e s p o n s e d e f i c i t * c a t a r a c t s * c y t o t o x i c T c e l l d e f i c i e n c y b y a p o p t o s i s v i a b l e / u n d e r s t u d y * * behavioral defects, under study * i n f l a m m a t i o n d e f i c i t , v W F a n d p l a t e l e t d e f i c i e n c y not O-glycan specific *

34. Branch Specificity in O-Glycan Structure-Function Relationships Core 2 O-Glycans Myeloid cell homeostasis and inflammation response Core 1 O-Glycans ST3GalT-I-dependent + CD8 T cell homeostasis b6 b3 Ser/Thr

35. N- and O-Glycans Modulate Physiologic Systems Glycosyltransferase and Glycosidase Specificity: Regulated Expression Competition with other Golgi Enzymes Different Substrate (protein?) Specificities Multiple isozymes