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: The mucins found as components of mucus gel layers at mucosal surfaces throughout the body play roles in protection as part of the defensive barrier on an organ and tissue specific basis. The human MUC gene family codes up to 20 known proteins, which can be divided into secreted and membrane-associated forms each with a typical protein domain structure. The secreted mucins are adapted to cross link in order to allow formation of the extended mucin networks found in the secreted mucus gels. The membrane-associated mucins possess membrane specific domains which enable their various biological functions as part of the glycocalyx. All mucins are highly O-glycosylated and this is tissue specific and linked with specific biological functions at these locations. Mucin biology is dynamic and the processes of degradation and turnover are well integrated with biosynthesis to maintain a continuous mucosal protection against all external aggressive forces. Interaction of mucins with microflora plays an important role in normal function. Mucins are modified in a variety of diseases and this may be due to abberant mucin peptide or glycosylation. Mucins represent a family of glycoprotein having fundamental roles in mucosal protection and communication with external environment. The review emphasises the nature of mucins as glycoproteins and their role in presenting an array of glycan structures at the mucosal cell surface.Biochimica et Biophysica Acta 05/2014; · 4.66 Impact Factor
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ABSTRACT: Tobacco smoke exposure, the major cause of chronic obstructive pulmonary disease (COPD), instigates a dysfunctional clearance of thick obstructive mucus. However, the mechanism underlying the formation of abnormally viscous mucus remains elusive. We investigated whether nicotine can directly alter the rheological properties of mucin by examining its physicochemical interactions with human airway mucin gels secreted from A549 lung epithelial cells. Swelling kinetics and multiple particle tracking were utilized to assess mucin gel viscosity change when exposed to nicotine. Herein we show that nicotine (≤ 50 nM) significantly hindered post-exocytotic swelling and hydration of released mucins, leading to higher viscosity, possibly by electrostatic and hydrophobic interactions. Moreover, the close association of nicotine and mucins allows airway mucus to function as a reservoir for prolonged nicotine release leading to correlated pathogenic effects. Our results provide a novel explanation for the maltransport of poorly hydrated mucus in smokers. More importantly, this study further indicates that even low concentration nicotine can profoundly increase mucus viscosity and thus highlight the health risks of second-hand smoke exposure.AJP Lung Cellular and Molecular Physiology 05/2014; · 4.04 Impact Factor
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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; · 4.60 Impact Factor