N-glycosylation enzyme Mpi is essential for mucin O-glycosylation, host-microbe homeostasis, Paneth cell defense, and metabolism.

Intestinal homeostasis relies on a protective mucus layer that separates bacteria from the host, with Muc2 as its primary component. This secreted, gel-forming mucin is heavily O-glycosylated, allowing it to retain water and support beneficial bacteria. For the first time, we demonstrate that Muc2 N-glycosylation plays a critical in mucin maturation, O-glycosylation, barrier integrity, and the prevention of dysbiosis. Using mouse models with global and intestine-specific N-glycan deficiency- caused by the loss of the mannose producing enzyme, we uncover an unexpected link between N-glycosylation and intestinal homeostasis. Our findings reveal that hypomorphic mice are highly sensitive to DSS-induced colitis, while ; mice spontaneously develop disease, exhibiting increased ER stress and dysbiosis. Additionally, electron microscopy, proteomics, and gene expression analyses of goblet and Paneth cells indicate immaturity, mitochondrial loss, and disruptions in lipid metabolism. These results highlight the fundamental role of N-glycosylation in maintaining intestinal homeostasis.