O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal ho-meostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial dif-ferentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell pro-teome. It was found that the fully differentiated butyrate -stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O- glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these pat-terns in homeostasis and dysbiosis at the gut-microbiota interface.

Automated summary

This study identified specific mucin O-glycans that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 using porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. The results showed that the fully differentiated butyrate-stimulated cells have a higher expression of sialylated O-glycan structures, while fucosylation is downregulated during differentiation. These findings provide insights into the dynamic O-glycosylation patterns in the gut and their functional role in gut-microbiota homeostasis and dysbiosis.