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Title: Investigating the supramolecular organisation of respiratory mucins in health and disease
Author: Hughes, Gareth
ISNI:       0000 0004 7657 6786
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2018
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The polymeric mucins, MUC5B and MUC5AC provide the structural and functional backbone of the respiratory mucus barrier, conferring both viscoelastic and antimicrobial properties onto these complex secretions. Within the mucus barrier, polymeric mucins are believed to be highly organised, arranged in a network-like formation. Structural details regarding how mucins are packaged, secreted and expand to form a functional barrier remain elusive. In addition, despite recent investigations into mucin secondary structure, it is unclear as to how the heavy glycosylation found within mucin domains can influence polymer conformation. Through the use of electron microscopy techniques in combination with biophysical analyses we performed an in depth structural analysis on purified MUC5B and MUC5AC, we then utilised these same techniques to investigate mucin packaging and expansion in response to changes in calcium concentration and pH levels. Finally through structural investigations into the native organisation of the salivary mucus barrier we aimed to investigate the hierarchical mucin topology present within mucosal systems, thus enabling us to draw conclusions about how this organisation may be compromised in disease states such as Cystic Fibrosis (CF). Through transmission electron microscopy (TEM) combined with single particle analysis, we provide evidence to suggest that MUC5B contains bead-like structures, which repeat along the polymer axis and suggest that MUC5B may consist of repeating motifs derived from distinct glycosylation patterns, rather than the disorganised random coil conformation previously assumed. Moreover, through TEM and rate zonal centrifugation, we demonstrate that respiratory mucins form highly entangled linear polymers, the structures of which are sensitive to calcium concentration and fluctuations in pH. In the presence of Ca2+ at pH 5.0, MUC5B adopted a compact conformation which was lost upon removal of calcium, via EGTA, and through increasing the pH to 7.4. We further suggest that the expansion of mucins following secretion occurs via two-step process, with an initial rapid expansion followed by a slower protease-independent maturation process, which is driven by distinct changes to the mucin interactome. Through isolation of mucins derived from CF sputum we have assessed the structural and biophysical changes that occur to the mucin network during CF progression, we suggest that the CF mucin network is severely degraded, with mucin glycopeptides predominating. Furthermore, we suggest that poly (acetyl, arginyl) glucosamine (PAAG) may represent an attractive treatment for CF lung disease, here we highlight its ability to directly bind MUC5B and induce its linearisation, resulting in a less viscous mucin network. Here we provide novel insights into the structural organisation of MUC5B and MUC5AC on both intra- and intermolecular levels, and shed light onto the processes required for mucin packaging and expansion, which have allowed us to understand the mechanisms behind aberrant mucus transport in CF.
Supervisor: Ford, Robert ; Thornton, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Saliva ; Electron Microscopy ; Cystic Fibrosis ; Mucus ; MUC5B ; Mucin ; MUC5AC