Mpi-driven N-glycosylation orchestrates mucin O-glycosylation and intestinal homeostasis.

The intestinal mucus barrier physically separates the epithelium from the dense microbial community of the gut and is essential for intestinal homeostasis. The principal component, the gel-forming mucin MUC2, is extensively glycosylated, yet how different classes of glycans regulate mucin function remains unclear. Here we show that N-glycosylation is required for proper MUC2 maturation and mucus barrier integrity. Using mouse models with hypomorphic or intestinal epithelial-specific loss of the mannose-generating enzyme MPI, which is required for N-glycan synthesis, we find that impaired N-glycosylation disrupts mucin processing and secretion. MPI deficiency results in severe susceptibility to dextran sulfate sodium-induced colitis or spontaneous intestinal inflammation, accompanied by endoplasmic reticulum stress, microbial dysbiosis, and defects in Paneth cells. These findings demonstrate N-glycosylation is critical for mucus barrier function and reveal an unexpected link between N-glycosylation and intestinal inflammatory disease.