Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.
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 subdomains (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.
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Caroline
C
Ridley
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Nikos
N
Kouvatsos
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Bertrand D
BD
Raynal
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Marj
M
Howard
the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Richard F
RF
Collins
the INSERM U995, University of Lille, F-59045 Lille, France, and.
Jean-Luc
JL
Desseyn
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Thomas A
TA
Jowitt
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Clair
C
Baldock
the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27517-7248.
C William
CW
Davis
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Timothy E
TE
Hardingham
From the Wellcome Trust Centre for Cell-Matrix Research and the Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom, dave.thornton@manchester.ac.uk.
David J
DJ
Thornton
D002118:Calcium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D055257:Mucin-5B / Q000378:metabolism*; D012137:Respiratory System / Q000378:metabolism*; D014461:Ultracentrifugation
D055257:Mucin-5B; D002118:Calcium
Analytical Ultracentrifugation; Cystic Fibrosis; Goblet Cell; Mucin; Mucus; Recombinant Protein Expression; Single Particle Analysis