Unpacking a gel-forming mucin: A view of MUC5B organization after granular release

1Department of Biochemistry and Biophysics and Cystic Fibrosis/Pulmonary Research and Treatment Center, Chapel Hill, NC, USA.
AJP Lung Cellular and Molecular Physiology (Impact Factor: 4.08). 09/2009; 298(1):L15-22. DOI: 10.1152/ajplung.00194.2009
Source: PubMed


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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    • "Moreover, whether this model can be generalised to MUC5AC and MUC5B, which form linear rather than branched polymers, has yet to be determined. Electron microscopy images of salivary mucins suggest that intragranular MUC5B is not packaged as a random coil but exists as a cross-linked structure that is organised around 'nodes' containing the terminal protein domains of the mucin polypeptide (Kesimer et al., 2010). However , the involvement of Ca 2+ ions in these 'nodes' has not been determined. "
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    ABSTRACT: Our current understanding of cystic fibrosis (CF) has revealed that the biophysical properties of mucus play a considerable role in the pathogenesis of the disease in view of the fact that most mucus-producing organs are affected in CF patients. In this review, we discuss the potential causal relationship between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the production of mucus with abnormal biophysical properties in the intestine and lungs, highlighting what has been learned from cell cultures and animal models that mimic CF pathogenesis. A similar cascade of events, including mucus obstruction, infection and inflammation, is common to all epithelia affected by impaired surface hydration. Hence, the main structural components of mucus, namely the polymeric, gel-forming mucins, are critical to the onset of the disease. Defective CFTR leads to epithelial surface dehydration, altered pH/electrolyte composition and mucin concentration. Further, it can influence mucin transition from the intracellular to extracellular environment, potentially resulting in aberrant mucus gel formation. While defective HCO3(-) production has long been identified as a feature of CF, it has only recently been considered as a key player in the transition phase of mucins. We conclude by examining the influence of mucins on the biophysical properties of CF sputum and discuss existing and novel therapies aimed at removing mucus from the lungs.
    The international journal of biochemistry & cell biology 03/2014; 52. DOI:10.1016/j.biocel.2014.03.011 · 4.05 Impact Factor
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    • "The fact that Otogelin-like is glycosylated and able to form oligomers suggests that it is capable of forming the matrix of a mucus-like barrier over the surface of the embryo. We suggest that the Otogelin-like protein can undergo massive expansion once it is secreted to form a layer over the surface, just like mucins do (Kesimer et al., 2010). However, our knockdown experiments suggest that this Otogelin-like protein is not sufficient on its own to mediate anti-microbial defence of the epidermis and that there are likely to be other secreted molecules. "
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    ABSTRACT: The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.
    Development 03/2014; 141(7). DOI:10.1242/dev.102426 · 6.46 Impact Factor
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    • "The transport of polymeric mucins from the ER to the Golgi and through the Golgi has been little studied, but likely involves modulation of COP-II transport vesicle size to accommodate large cargoes as has been described for collagen (25). In the Golgi, Muc5b undergoes further polymerization in linear chains up to 20 monomers in length (3, 26). The structure of Muc5ac is less well studied, but appears to be more similar to Muc2 that forms branched polymers resulting in formation of a covalent net (9). "
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    ABSTRACT: Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3x10^6 D per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ~1 um in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among MARCKS, cysteine string protein (CSP), HSP70 and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of
    Frontiers in Endocrinology 09/2013; 4:129. DOI:10.3389/fendo.2013.00129
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