The Role of Defective CFTR Ion Transport on Mucin Sialylation and its Consequences on Mucus Physiology

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PROJECT SUMMARY Cystic Fibrosis (CF) is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an ion channel responsible for the transport of chloride and bicarbonate across the apical cell surface. CF affects multiple organs, and in the lungs, results in mucus stasis, chronic infection/lung damage, and mortality. Mucus stasis is a pathologic hallmark of CF, and many of the gel-forming properties of airway mucus are provided by the gel-forming mucins, MUC5B and MUC5AC. These gel-forming mucins are extensively O-linked glycosylated, and terminal sialylation of these glycans contributes to their negative charges and interaction with the ionic microenvironment established by CFTR. Changes in O-linked sialylation of gel-forming mucins would therefore be expected to alter their physiochemical characteristics. Early evidence shows that defective CFTR can reduce mucin sialylation, however, the mechanistic basis for this, its relationship to impaired CFTR anion transport, and its consequences on mucus function are unknown. Furthermore, low charged mucin glycoforms have been linked to pathologic mucus in multiple airway diseases, but the consequences of altered sialylation on mucin structure and mucus physiology have not been determined. Our preliminary data shows that sialyltransferase expression is downregulated in CF primary human bronchial epithelial cells, and that inhibiting sialylation in vitro and in vivo leads to impairment of mucociliary transport independent of mucus hydration. Through this proposed study, we will identify the role of CFTR anion transport on mucin sialylation and determine the functional consequences of altered mucin sialylation on mucin structure and mucus transport. Our overarching hypothesis is that impaired CFTR ion transport downregulates the sialylation of mucins, which decreases their negative charge, resulting in mucin structural abnormalities and mucus stasis in CF. To test this, we have developed two specific aims. In aim 1, we will determine the effect of CFTR anion transport on mucin sialylation by pharmacologically inhibiting or restoring anion transport in vitro and in vivo, followed by assessing changes in sialylation by quantifying sialyltransferase expression, sialyltransferase protein levels, and mucin sialylation. In aim 2, we will directly downregulate or upregulate global or specific sialyltransferase activity and assess the changes in MUC5B or MUC5AC electrostatic properties, conformation, and physiology to define the consequences of altered sialylation on gel forming airway mucins. These studies will advance the field by revealing a new mechanism of mucus stasis in CF, while identifying novel therapeutic targets for treatment of CF and possibly other diseases of mucus stasis.