Decoding the Cellular Glycome: Mucin-type O-glycans in Health and Disease Restricted; Files Only

Kudelka, Matthew Robert (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/xg94hq37z?locale=en
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Abstract

Cells are coated with a glycocalyx rich in carbohydrates, or glycans, that facilitate interactions of cells with the environment. Mucin-type O-glycans are a major class of cell surface glycosylation and are critical for embryogenesis and altered in a range of diseases, such as inflammatory bowel disease (IBD) and cancer. O-glycan synthesis is initiated in the Golgi with addition of GalNAc to serine or threonine in a glycoprotein and further extended by various monsaccharides to form complex, but poorly defined, structures. We sought to define the normal O-glycome, evaluate how it changes in disease, and determine the consequences of those changes.

Traditional approaches for O-glycomics chemically release a finite number of glycans from a sample in an inefficient and degradative process. To address this, we developed a technology termed Cellular O-glycome Reporter/Amplification (CORA) that amplifies and sequences the O-glycome from cells, yielding ~100-1000x increased sensitivity over traditional approaches. We incubated cells with a peracetylated GalNAc-Bn chemical O-glycan precursor that is taken up into cultured cells and modified by endogenous glycosyltransferases utilizing native sugar donors to form Bn-O-glycans reprenting the native repertoire of cellular O-glycans, followed by secretion, purification, and analysis. We combined our next-generation glycomics across a range of cell types with computational models to predict the size of the human glycome and have begun to define the normal cellular O-glycome and how it changes in disease.

IBD arises from a disrupted immune response to the gut microbiota in genetically susceptible individuals. Recent evidence indicates that the gut microbiota is spatially disrupted in IBD with more prominent dysbiosis in the mucosa versus the luminal contents. O-glycans are the major class of glycosylation in the intestinal epithelia and overlying mucus layer and are truncated in IBD with loss of terminal epitopes and increased expression of simple sugars such as GalNAc (Tn), and Sialyl Tn (STn). Cosmc is a molecular chaperone on the X-chromosome required for normal extension of O-glycans beyond the simple Tn and STn antigens. A recent GWAS implicated Cosmc as a sex-specific risk gene in IBD. To explore the role of Cosmc and altered glycosylation in IBD, we deleted Cosmc in the intestinal epithelia and observed loss of the mucus layer, enhanced bacterial-epithelial, and ultimately spontaneous microbe-dependent inflammation. KO mice exhibited a loss of microbial diversity and dysbiosis in the colonic mucosa but not in the overlying lumen or small intestine resembling the spatial disruption of the gut microbiota in IBD. In contrast to male mice with loss of one allele of Cosmc, female mice with loss of one allele were completely protected from spontaneous colitis and partially protected from experimental colitis due to lateral migration of WT mucin glycocalyx over KO cells. These results validate Cosmc as a sex-specific risk gene that spatially regulates the microbiota and links IBD disease genetics, altered glycosylation, and spatial disruption of the gut microbiota. Collectively, these studies define the normal O-glycome, how O-glycans change in disease, and how these changes contribute to disease biology.

Table of Contents

Table of Contents

Chapter 1. An Introduction: Mucin-type O-glycans in Cancer………………………1

Chapter 2. An Introduction: Epithelial Glycosylation in Inflammatory Bowel Disease…………………………………………………………………………………...73

Chapter 3. Cellular O-Glycome Reporter/Amplification to Explore O-Glycans of Living Cells………….…………………………………………………………………..92

Abstract……………………………………………………………………………....93

Introduction…………………………………………………………………………..94

Results…………………………………………………………………………….….95

Cells uptake Ac3GalNAc-α-O-Bn and secrete Bn-O-glycans

Synthesis of Core 1-based Bn-O-glycans requires T-synthase

Accuracy of CORA

Sensitivity of CORA

Profiling the O-glycome of mouse and human primary cells

CORA promotes the discovery of novel glycans

CORA can evaluate the complexity of the cellular O-glycome

Discussion…………………………………………………………………………..108

Methods…………………………………………………………………………….110

Supplemental Information………………………………………………………….118

Chapter 4. Cosmc is an X-linked inflammatory bowel disease risk gene that spatially regulates gut microbiota and contributes to sex-specific risk……..………………..146

Abstract……………………………………………………………………………..147

Significance Statement………...……………………………………........................148

Introduction…………………………………………………………………………149

Results………………………………………………………………………………152

Spontaneous inflammation in male KO mice

No spontaneous inflammation in female mosaic mice

Microbes drive inflammation

Regional changes in the microbiota

Discussion…………………………………………………………………………..163

Methods………………………………….………………….………………………165

Supplemental Information…………………………………….……………………168

Chapter 5. Discussion…………………………………………………………......…..176

References……………………………………….……………………………………..190


Figure and Table Index

Chapter 1.

1. Figure 1.1. Diverse glycoconjugates of the cell.

2. Figure 1.2. Tumor-associated carbohydrate antigens.

3. Figure 1.3. The biosynthesis of O-GalNAc-type O-glycans.

4. Figure 1.4. The role of Cosmc in core 1 O-glycan synthesis.

5. Table 1.1. Truncated O-glycans in cancer.

6. Table 1.2. Altered terminal O-glycans in cancer.

7. Table 1.3. Frequency of altered Tn, STn, T, SLea, and SLex in cancer.

8. Table 1.4. Serum Biomarkers.

9. Table 1.5. Tumor imaging.

Chapter 3.

10. Figure 3.1. Overview of CORA.

11. Figure 3.2. The chaperone Cosmc and active T-synthase are required for production of core 1- and core 2-based Bn-O-glycans.

12. Figure 3.3. Accuracy of CORA for profiling the O-glycome.

13. Figure 3.4. Sensitivity of CORA.

14. Figure 3.5. MALDI-TOF-MS/MS profiling of the O-glycome of primary cells.

15. Supplementary Figure 3.1. MALDI profiles of compounds used in this study.

16. Supplementary Figure 3.2. Bn-O-glycans produced by HEK293 and Molt-4 cells.

17. Supplementary Figure 3.3. Bn-GalNAc peracetylation increases CORA sensitivity.

18. Supplementary Figure 3.4. Stability of acetylation in complete media.

19. Supplementary Figure 3.5. Optimizing concentration of Ac3GalNAc-Bn and incubation time.

20. Supplementary Figure 3.6. CORA does not alter cell viability.

21. Supplementary Figure 3.7. CORA does not alter cell morphology or granularity.

22. Supplementary Figure 3.8. CORA does not alter cell surface O-glycosylation.

23. Supplementary Figure 3.9. CORA does not alter cell surface N-glycosylation.

24. Supplementary Figure 3.10. Stability of O-glycans in media with cells.

25. Supplementary Figure 3.11. Evaluation of potential Bn-glycans produced from cells incubated with Ac3GlcNAc-Bn.

26. Supplementary Figure 3.12. Accuracy of CORA for profiling the O-glycome.

27. Supplementary Figure 3.13. MALDI-TOF/TOF-MS/MS analysis of permethylated O-glycans derived from WEHI-3 cells from Fig. 3.4a.

28. Supplementary Figure 3.14. MALDI-TOF/TOF-MS/MS analysis of permethylated O-glycans derived from HL-60 cells.

29. Supplementary Figure 3.15. Reproducibility of CORA.

30. Supplementary Figure 3.16. Analysis of HUVEC, MKN45, and WEHI-3 O-glycans by ESI-MS.

31. Supplementary Figure 3.17. Transcript levels of Core 2 GnT1-3 in cell lines.

32. Supplementary Figure 3.18. Sensitivity of CORA.

33. Supplementary Figure 3.19. O-glycome of murine pulmonary endothelial cells.

34. Supplementary Figure 3.20. Profiling the O-glycome of Primary Cells.

35. Supplementary Figure 3.21. MALDI-TOF/TOF-MS/MS analysis of permethylated O-glycans derived from HUVEC cells from Fig. 3.5.

36. Supplementary Figure 3.22. MALDI-TOF-MS profiling of the O-glycome of primary human dermal fibroblasts.

37. Supplementary Figure 3.23. Glycan frequency across cell lines evaluated in CORA.

38. Supplementary Figure 3.24. Computational model to estimate the size of the non-sulfated human cellular O-glycome.

39. Table 3.1. Information on cell lines and primary cells, the MS analyses, and data presented in the figures.

40. Table 3.2. Summary of glycan masses and compositions observed in the cell lines.

41. Table 3.3. Glycan structures from three cell lines determined by MS/MS.

Chapter 4.

42. Figure 4.1. Characterization of IEC-Cosmc mice.

43. Figure 4.2. Spontaneous inflammation in IEC-Cosmc-KO but not mosaic mice.

44. Figure 4.3. DSS colitis in KO and mosaic mice.

45. Figure 4.4. Microbes drive inflammation in KO mice.

46. Figure 4.5. Cosmc regionally regulates the gut microbiota community structure.

47. Supplementary Figure 4.1. Breeding strategy and characterization of IEC-Cosmc mice.

48. Supplementary Figure 4.2. Time course of clinical inflammation.

49. Supplementary Figure 4.3. Effects of cohousing and rectal prolapse on inflammation in KO mice.

50. Supplementary Figure 4.4. Hyperplasia in KO mice but not mosaic mice.

51. Supplementary Figure 4.5. Bacteria-epithelial interactions in the distal colon.

52. Supplementary Figure 4.6. Bacterial counts of mice treated with antibiotics.

53. Supplementary Figure 4.7. Analysis of fecal IgA.

54. Supplementary Figure 4.8. Cosmc controls diverse host pathways that regulate the microbiota and inflammation in the distal colon.

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