Role of Cosmc and Tn Antigen in T Cell Biology and the O-Glycoproteome Open Access

Cutler, Christopher (Summer 2019)

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T lymphocytes are indispensable to the adaptive immune system and proper execution of their role requires appropriate glycosylation. Despite evidence that O-glycosylation of serine and threonine residues in glycoproteins is dynamically and temporally regulated during T cell maturation and activation, little is known about how specifically O‑glycosylation impacts T cells or even which proteins on T cells are modified with O‑glycans. Here, I present my findings that extended O‑glycans beyond the Tn antigen are important for T cell homing to secondary lymph organs and survival in the periphery. These findings underscore the importance of O‑glycosylation in regulating T cell biology and emphasize the need for ongoing attention to glycosylation in studies of T cell development and function. Additionally, I present a method for sensitive identification of Tn-expressing glycosylated O‑glycoproteins, providing proof of concept for future targeted proteomics to identify proteins with O‑glycans, which will allow for the exploration of the physiological effects of glycosylation.

Table of Contents

Chapter 1: Background and Significance 1

Introduction 1

1.1 T cells 3

1.1.1 T cell maturation 3

1.1.2 Function of mature T cells 5

1.2 Glycoproteins 8

1.2.1 Protein glycosylation 9

1.2.2 Detecting glycoproteins 14

1.2.3 Glycoprotein linked pathology 19

1.3 T cell glycosylation 24

1.3.1 Lineage commitment and thymic selection 24

1.3.2 Signaling in mature T cells 28

1.3.3 T cell homing 32

1.3.4 T cell recognition of glycan-dependent antigens 33

1.4 Summary 36

1.5 Tables and Figures 38

Chapter 2: Cosmc is required for T cell persistence in the periphery 40

2.1 Abstract 41

2.2 Introduction 42

2.3 Results 44

2.3.1 Lck-Cre drives T cell specific knockout of Cosmc 44

2.3.2 Cosmc KO results in dramatically reduced T cell numbers 46

2.3.3 Cosmc KO does not block thymocyte maturation 48

2.3.4 TCKO T cells are deficient in homing to SLO 50

2.4 Discussion 51

2.5 Materials and Methods 59

2.6 Tables and Figures 63

Chapter 3: A Sensitive Glycoproteomic Method for Identifying O-glycosylated Proteins Using Low Sample Quantities 73

3.1 Abstract 74

3.2 Introduction 75

3.3 Results 77

3.3.1 Recombinant ST6GalNAc1 is active, accepts biotin-linked CMP-Neu5Ac, and acts to autosialylate itself 77

3.3.2 Truncation of ST6GalNAc1 80

3.3.3 Enzymatic activity of ST6GalNAc1-trunc on glycopeptides 82

3.3.4 Activity of ST6GalNAc-1-trunc on glycopeptides 83

3.3.5 ST6GalNAc1-trunc acts effectively on cells and cell lysates 84

3.4 Discussion 85

3.5 Materials and Methods 88

3.6 Tables and Figures 94

Chapter 4: Discussion and future directions 110

4.1 Introduction 111

4.2 Unique T cell requirement of O-glycans 111

4.3 Lymphocyte homing 113

4.4 T cell signaling and survival 114

4.4 Cancer glycobiology 115

4.5 Glycoproteomics 117

4.6 Conclusion 119

References 121

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