(382) Mechanisms of Cysteine-Mediated Polymerization and Secretion of Muc5ac and Muc5b

Background: Mucociliary clearance (MCC) is required for respiratory defense. In health, mucus is thin and easily transported by MCC. In cystic fibrosis (CF), mucus is thick and poorly transported. Consequently, key pulmonary manifestations of CF are MCC impairment, mucus aggregation, and airway inflammation. Mucus dysfunction is not adequately treated with existing therapies. It is thus essential to improve our mechanistic understanding of mucin biology. Two polymeric mucins, MUC5AC and MUC5B, form the matrix of airway mucus and are critical determinants of mucus function in health and dysfunction in CF.

During synthesis, polymeric mucins assemble via covalent disulfide bonds through their conserved carboxy (C-) and amino (N-) terminal domains. We previously showed that non-selectively reducing disulfide bonds in mouse lungs improves mucus viscoelasticity, airflow, and MCC. Accordingly, we hypothesize that determining specific mucin disulfide polymerization and mucin transport mechanisms could identify selective strategies for preventing mucus dysfunction.

Methods: We transfected HEK293 cells with plasmids encoding Muc5ac and Muc5b N- and C- terminal domains, where conserved cysteines predicted to mediate disulfide assembly have been mutated. We then validated these findings in full-length mucins in vivo, by making CRISPR/Cas9 gene-edited mice with point mutations in the same cysteines. To determine mechanisms for secretory transport of nascent mucin polymers between the endoplasmic reticulum (ER) and the Golgi, we performed knockdown studies to inhibit MIA3, an ER protein that transports cargo that is too large for typical machinery.

Results: Our results show that mutating Muc5ac C-terminal domain cysteines C3378 and C3380 abolished dimerization and its ability to properly traverse the secretory pathway. By contrast, mutating C3378, C3380 and C3418 resulted in abundant dimers in bronchoalveolar lavage fluid. This suggests that when only C3418 is present, Muc5ac is misfolded in the ER and undergoes a potential degradation pathway. However, when neither cysteine is available for dimerization in the ER, Muc5ac monomers traverse as such to the Golgi where they dimerize and get secreted.

Furthermore, mutating C1137 in the Muc5ac N-terminal domain resulted in the secretion of various sizes of Muc5ac polymers, from dimers to full-length polymers. Mutating C1177 has no effect on Muc5ac polymerization, but mutating C1137 and C1177 resulted in dimers, suggesting that Muc5ac still dimerized in the ER and was secreted despite not fully polymerizing in the Golgi. In both N- and C-terminal mutant animals, Muc5b assembly and secretion appeared unaltered, suggesting that the Muc5ac mutation could be exploited therapeutically for mucin isoform selective targeting.

In vitro data of Muc5b and Muc5ac showed similar results, suggesting that Muc5b and Muc5ac polymerize via conserved pathways. In vivo studies for Muc5b are ongoing.

Lastly, we have now also identified MIA3 as a novel component in the mucin assembly pathway. We found that MIA3 is expressed in goblet cells, and it is required for MUC5AC secretion in a human cell line. Additional studies are being performed to better understand the expression and function of MIA3 in mucin polymer assembly and secretion.

Conclusions: Collectively, our findings suggest that targeting mucin assembly or non-conventional trafficking could be used to achieve mucin-selective inhibition in CF.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (JARAMI20F0).

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