(404) Why are mucins so gigantic and is it rational to sever them to aid mucus clearance?

Background: MUC5B is a key macromolecular component of airway mucus and its absence causes lung pathologies associated with defective mucus clearance in both mice and humans. Mucins have unique, gel/elastic-like biophysical properties that support effective mucus transport. These properties are predicted to be dependent on mucin concentration, polymeric structure, and size, which reflect formation of intra- and inter-molecular disulfide bonds across specific mucin domains. Why mucins have evolved to such size ( >100 MDa) and whether reducing their size has therapeutic value is currently unknown.

Methods: Using CRISPR/Cas9 mutagenesis, we generated a mouse with two Cys/Ala substitutions (Cys1128, Cys1170) in Muc5b N-terminal D3 domain. WT, heterozygous mutant (Het), and homozygous mutant (KI) littermates where phenotyped using a variety of assays including tracheal and nasal mucociliary clearance (MCC), bronchoalveolar lavage (BAL) differential cell counts, airway mucus mass spectrometry and western blot, and histology. A suite of biophysical assays was used to characterize mucus harvested from WT, Het, and KI mouse tracheal epithelial cell ALI cultures (mTEC), including atomic force microscopy, light scattering, microrheology, microtribology, and microscopic measurements of horizontal vs. vertical mucus transport.

Results: Cys1128 and Cys1170 Ala substitutions resulted in selective production of Muc5b dimers in KI mice. Het mice produced Muc5b multimers shorter (≤8mers) than WT mice, suggesting a dominant negative “capping” on higher order multimerization. Quantitation of total Muc5b by western blot and mass spectroscopy demonstrated higher Muc5b recovery in BAL from Het and KI vs. WT mice. RNA expression levels for Muc5b were similar across genotypes, suggesting the intrapulmonary mucus accumulation reflected defects in clearance, not increased production. Consistent with this notion, Het and KI mice exhibited defective nasopharyngeal and tracheal MCC, and KI mice exhibited focal intrapulmonary mucus accumulation at 6 weeks of age. Both Het and KI mice exhibited nasopharyngeal hair/mucus plugging which likely caused sporadic respiratory distress and death in mice older than 3 months. BAL cell differentials were equivalent across genotypes. Using a novel conditionally reprogrammed cell-based method, WT, Het, and KI polarized mTEC were generated that produced Muc5b isoforms indistinguishable from their in vivo counterparts. Atomic force microscopy confirmed the exclusive presence of “short” dimers in KI mTEC mucus, with ~3 MDa MW and ~50 nm radius of gyration as measured by static light scattering. Complex viscosity measured by microrheology was reduced in both Het and KI mTEC secretions, and KI mTEC exhibited significant reductions in surface adhesive strength vs. WT. Preliminary data indicated that KI mTEC mucus was unable to support vertical mucus transport, suggesting its elastic modulus was insufficient for transport against gravity.

Conclusions: These data suggest that “giant” mucin multimers are required for normal airway mucus transport. Biophysical and modeling data predict that high-order multimeric mucins are required to achieve the overlap concentrations (interpenetration) necessary to generate the elastic modulus needed for mucus transport. Therapeutic reduction of mucin multimers may have opposing effects in the treatment of muco-obstruction: 1) reduced mucus elasticity can degrade ciliary clearance; 2) reduced mucus viscosity is predicted to improve cough clearance.

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