[Thesis]. Manchester, UK: The University of Manchester; 2018.
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.