PROJECT SUMMARY In many lung diseases--including asthma--excessive mucus disrupts clearance and obstructs airflow, but mucus dysfunction is not effectively treated by existing therapies. The chief macromolecules in airway mucus are MUC5AC and MUC5B. To study them causatively, we made Muc5ac and Muc5b knockout mice. We discovered that Muc5b is required for mucociliary clearance (MCC) in health, but Muc5ac is dispensable. Instead, Muc5ac causes mucus plugging and is required for airway hyperreactivity (AHR) in models of asthma. Nonetheless, despite its homeostatic requirements in mice and humans, we now know that excessive MUC5B/Muc5b can itself be detrimental in lung fibrosis (PF). These findings show significance, but they also highlight the need to find ways to prevent mucus dysfunction while also preserving defense. We postulate that this can be accomplished in part through an improved understanding of MUC5AC and MUC5B assembly mechanisms. MUC5AC/Muc5ac and MUC5B/Muc5b are very large proteins that form even larger polymers via linkages between their carboxyl (C-) and amino (N-) termini. Their pathologic properties depend on covalent disulfide bonds whose reduction reverses AHR and improves MCC, suggesting that therapeutic intervention targets could be revealed by determining precisely how mucins assemble. Mucin C- and N-terminal polymerization domains are homologous with the protein von Willebrand Factor (VWF). Cysteines required for VWF assembly are conserved in mucins. We have identified which are required for airway mucin assembly and function. Here we seek to continue determine structural requirements and cellular mechanisms for mucin polymerization and function. Emerging data suggest that a cluster of three cysteines referred to as a “cysteine triad” is crucial. One of member of the triad is the cysteine that forms an inter-molecular disulfide. This requires the acidic environment provided in the Golgi to facilitate disulfide exchanges that liberate cysteines needed for intermolecular disulfides. Low pH also protonates histidines in domains that surround the triad. The cysteine triads and pH-sensing histidines in VWF are conserved in mucins. We hypothesize that airway polymeric mucin biosynthesis is regulated by Golgi specific mechanisms that mediate mucus function and dysfunction. We will test this by 1) examining how assembly is regulated by Golgi-localized cysteine-triad dependent mechanisms, 2) determining the conserved histidine residues are required for pH-dependent N-terminal assembly, and 3) demonstrating how these affect mucin polymerization airway mucus functions. Studies here will advance the mucin biology field while laying groundwork for approaches to prevent mucus dysfunction while preserving defense. While research here focuses on asthma, results will have implications for diseases such as chronic obstructive pulmonary disease (COPD), CF, and other conditions where mucus dysfunction is prevalent.