(A) Topography of the normal colonic mucus layer. Bacteria in the outer mucus layer overlay the inner mucus layer, which is normally impenetrable to bacteria; DAPI (blue), MUC2 (green; sc-15334, H-300), bacteria (16S FISH; red), magnification: 100x15. (B) The MUC2 glycoprotein has an N-terminal trimerization and a C-terminal dimerization domain with a PTS domain that is rich in proline, threonine, serine in between. The N-terminus contains three von Willebrand D domains and the C-terminus contains one (not shown). The PTS domain is highly O-glycosylated in the Golgi and interspersed by two CysD domains, contributing to intramolecular disulfide bonds and intermolecular non-covalent interactions. (C) MUC2 is synthesized in the endoplasmic reticulum, N-glycosylated and dimerized. It is then transported to the Golgi through an N-glycan-dependent mechanism, where it is O-glycosylated and forms trimers. (D) MUC2 is released and unpacked in the inner mucus layer through alkalinization and chelation of Ca2+ ions and then further unpacked in the outer mucus layer through proteolysis; the red line indicates cleavage by host proteases.