1. O-Glycans

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

3. Mucins are heavily O-glycosylated

4. Mucin Production in the Intestinal Crypts

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

6. Core 1 and Core 2 O-Glycan Synthesis

8. Core 3 and Core 4 O-Glycan Synthesis

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

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. 0 40 60 80 100 120 20 0 1 2 3 4 cells/ml x10 3 Core 2 GlcNAcT Deficiency Results in Leukocytosis WBC cells/ml x10 3 Neu Lym EosMon wt/∆ wt ∆/∆

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

14. 50 100 150 200 250 300 350 400 500 450 Time (hrs.) Post-Thioglycollate Stimulus Impaired Neutrophil Recruitment to the Peritoneum of Mice Lacking Core 2 GlcNAcT 550 600 wt null Neut. # x10 4 042

15. -/- wt/wt -/- wt/wt ∆/∆ wt/wt -/- wt/wt -/- wt/wt wt/wt-/- Neutrophil Recruitment in a model of peritonitis 100 %WT Defic.P-SelL-SelE-SelP/EFT7C2-1 0 40

16. 0 2 4 6 8 10 12 14 ∆/∆ wt/wt Normal Leukocyte Abundance and Homing in Lymph Nodes of Mice Lacking the Core 2 GlcNAcT Gene 0 1.5 1.0 0.5 ∆/∆ wt/wt PLNMLN PP Cells x10 6 wt null MNPNPP CMFDA+ cells

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

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

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

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

23. 100X 200X WTST3Gal-I Null PNA Binding (brown) in Thymus

24. Thymic CD8-MHC Binding Modulated by ST3Gal-I Cortex Medulla SS SS CD8   MHC I  3 protruding loop

25. Thymic CD8-MHC Binding Modulated by ST3Gal-I Cortex Medulla SS SS CD8   MHC I  3 protruding loop

26. Cell # % of WT 100 thymusspleen 50 bloodlymph nodes 25 CD4CD8 75 Deficiency of Peripheral CD8 + T Cells in ST3Gal-I Null Mice

27. 0 20 40 100 60 80 % Annexin V+ Time (hrs) - 024 1B11 024 0 Immune Activation Blocks CD8 + T Cell Apoptosis in ST3Gal-I Null Mice anti-CD3 pretreatment wt ∆/∆ 1B11

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

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

30. 75 50 25 0 0612244872 % PNA high Hours post-TCR Activation 100 Core 1 and Core 2 O-Glycans in CD8 + T Cell Activation % 1B11 high

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

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

33. ocytosis, lymphoidhomingdefect, and inflammatoryresponse deficit cataracts cytotoxic T cell deficiency by apoptosis viable/under study inflammation deficit, vWF and platelet deficiency Induced O-glycan Deficiencies in the Mouse and Biological Effects not O-glycan specific * * * * * * * * * behavioral defects, under study

34. Branch Specificity in O-Glycan Structure-Function Relationships Core 1 O-Glycans ST3GalT-I-dependent Core 2 O-Glycans CD8 + T cell homeostasis Myeloid cell homeostasis and inflammation response 33 66 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