Structure of the Disulfide-Rich Mucin CysD Module and Its Contribution to the Formation of Mucus Hydrogels

The hundreds of square meters of internal epithelia in the respiratory and gastrointestinal tracts are protected from physical and biological threats by hydrogels of enormous proteins known as gel-forming mucins. The intestinal mucin Muc2 is more than 5000 amino acids long and is modified by thousands of O-linked glycans. The Muc2 protein assembles by disulfide-mediated cross-linking into ultra-long polymers, which further associate with one another non-covalently, in a pH- and calcium-dependent manner, during formation of the protective network. We observe that a module of about 100 amino acids containing 5 disulfide bonds mediates non-covalent interactions in Muc2. This module, the CysD domain, appears in multiple copies within mucin proteins. Respiratory and gastrointestinal mucins both contain CysD domains, but they have diverged in the number of copies of these modules and in the sequences outside the conserved cysteine pattern. These differences may underlie the distinct rheological properties and functions of mucins in these two physiological contexts (i.e., hosting the microbiome and shielding the epithelium in the gut vs. sweeping the cell surface clean of particulate matter and pathogens in the airways). To better understand the contribution of CysD domains to mucin assembly, we are in the process of determining the structure of a CysD domain from Muc2. From the CysD structure, we aim to determine how the domain contributes to pH-dependent protein-protein interactions. Mucin assembly is crucial for its function as a hydrogel, and understanding the assembly mechanism might suggest means to stabilize the network and enhance barrier function.