Assembly and proteolytic cleavages of gel-forming mucins

Abstract: Gel-forming mucins are large glycoproteins constituting the main structural component of themucus layer protecting epithelial surfaces of the body. This thesis aims to a deeper understandingof how gel-forming mucins are synthesized and assembled, and how they may interact with othermolecules. This knowledge is fundamental for understanding the physiology of the mucus barrierin health and disease.That mucins are large, heterogeneously glycosylated and forms polymeric structures,make them hard to study by conventional methods. Our approach has been to express parts of theproteins separately in different cells allowing biochemical analyses.When expressing the N-terminal part of the human MUC2 mucin the protein formeddisulfide-linked oligomers late in the secretory pathway. The secreted oligomers were heldtogether in a trimeric trypsin-resistant core fragment. That the protein formed trimers wasconfirmed by electron microscopy. Expression of the C-terminal region of the mucin showed thatthis part formed disulfide-linked dimers in the endoplasmic reticulum. The dimeric nature wasconfirmed by electron microscopy.When studying the C-terminal part of MUC2, a cleavage between an aspartate and aproline located in a GDPH sequence was observed. This cleavage was shown to be a slow, nonenzymaticprocess occurring at a pH below 6. The cleavage could occur in the later more acidicparts of the secretory pathway as neutralization of these compartments inhibited the cleavage.In vitro studies showed that the cleavage generated a new reactive C-terminus that could link theprotein to other molecules. The human MUC5AC mucin also contains a GDPH sequence andshows high sequence similarities to MUC2 in the area surrounding this sequence. That thecleavage also occurred in MUC5AC was shown after expressing its C-terminal part. Although thecleavage occurred already in the neutral endoplasmic reticulum further cleavage was observed atan acidic pH. Also after this cleavage a reactive C-terminus was formed. The generation of thereactive ends of these mucins can have implications in diseases such as cystic fibrosis andasthma, both states characterized by mucus pluggings and lower pH in the mucus of the airways.The lower pH could lead to increased cleavage causing cross-links between the mucins or tomolecules found on the epithelial surface thereby adding to the mucus retention seen in thesediseases.For parasites to reach the epithelial surfaces they have to penetrate the mucus gel.Entamoeba histolytica, the parasite causing amoebiasis secretes cysteine proteases that aresuggested to degrade the colonic mucus, mainly composed of MUC2. To test this hypothesis, therecombinant parts of MUC2 and insoluble mucins prepared from LS 174T cells were treated withproteases secreted by the amoeba. The results showed that the cysteine proteases degraded themucus gel by targeting the C-terminal part of MUC2. This is, to our knowledge, the firstidentification of a specific cleavage mechanism, used by an enteric pathogen, to disrupt thepolymeric nature of the mucin gel.