Unpacking a gel-forming mucin: a view of MUC5B organization after granular release
ABSTRACT Gel-forming mucins are the largest complex glycoprotein macromolecules in the body. They form the matrix of gels protecting all the surface epithelia and are secreted as disulfide-bonded polymeric structures. The mechanisms by which they are formed and organized within cells and thereafter released to form mucus gels are not understood. In particular, the initial rate of expansion of the mucins after release from their secretory granules is very rapid (seconds), but no clear mechanism for how it is achieved has emerged. Our major interest is in lung mucins, but most particularly in MUC5B, which is the major gel-forming mucin in mucus, and which provides its major protective matrix. In this study, using OptiPrep density gradient ultracentrifugation, we have isolated a small amount of a stable form of the recently secreted and expanding MUC5B mucin, which accounts for less than 2% of the total mucin present. It has an average mass of approximately 150 x 10(6) Da and size Rg of 150 nm in radius of gyration. In transmission electron microscopy, this compact mucin has maintained a circular structure that is characterized by flexible chains connected around protein-rich nodes as determined by their ability to bind colloidal gold. The appearance indicates that the assembled mucins in a single granular form are organized around a number of nodes, each attached to four to eight subunits. The organization of the mucins in this manner is consistent with efficient packing of a number of large heavily glycosylated monomers while still permitting their rapid unfolding and hydration. For the first time, this provides some insight into how the carbohydrate regions might be organized around the NH(2)- and COOH-terminal globular protein domains within the granule and also explains how the mucin can expand so rapidly upon its release.
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ABSTRACT: Understanding how the body's natural defenses function to protect the oral cavity from the myriad of bacteria that colonize its surfaces is an ongoing topic of research that can lead to breakthroughs in treatment and prevention. One key defense mechanism on all moist epithelial linings, such as the mouth, gastrointestinal tract and lungs, is a layer of thick, well-hydrated mucus. The main gel-forming component of mucus are mucins, large glycoproteins that play a key role in host defense. This study focuses on elucidating the connection between MUC5B salivary mucins and dental caries, one of the most common oral diseases. Dental caries are predominantly caused by Streptococcus mutans adherence and biofilm formation on the tooth surface. Once S. mutans adheres to the tooth, it produces organic acids as metabolic byproducts that dissolve tooth enamel, leading to cavity formation. We utilize colony forming units and fluorescent microscopy to quantitatively show that S. mutans attachment and biofilm formation is most robust in the presence of sucrose and that aqueous solutions of purified human MUC5B protect surfaces by acting as an anti-biofouling agent in the presence of sucrose. In addition, we find that MUC5B does not alter S. mutans growth and decreases surface attachment and biofilm formation by maintaining S. mutans in the planktonic form. These insights point to the importance of salivary mucins in oral health and lead to a better understanding of how MUC5B could play a role in cavity prevention or diagnosis.Applied and Environmental Microbiology 10/2014; DOI:10.1128/AEM.02573-14 · 3.95 Impact Factor
- 05/2014; 59(5):788-789. DOI:10.4187/respcare.03319
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ABSTRACT: Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein-site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3 domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N-terminus (D1D2D′D3; NT5B) and sub-domains (D1, D1-D2, D2-D′-D3 and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide-linkage between D3 domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel forming polymeric mucins.Journal of Biological Chemistry 04/2014; jbc.M114.566679. · 4.60 Impact Factor