Decoding the Cellular Glycome: Mucin-type O-glycans in Health and Disease Public
Kudelka, Matthew Robert (2017)
Published
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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