Mucin Glycosylation and Sulphation in Airway Epithelial Cells Is Not Influenced by Cystic Fibrosis Transmembrane Conductance Regulator Expression

Abstract

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/ 5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (⌬F508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation.

Glycosylation of sputum mucins is altered in cystic fibrosis patients

“…This is in contrast to previous investigations showing that the glycosylation of pure secretions from submucosal glands (Schulz et al 2005) and epithelial tissue culture (Holmen et al 2004;Leir et al 2005) cannot distinguish between CF and non-CF. This therefore suggests that there is infection/inflammation-dependent glycosylation of epithelial secretions in CF, which can even affect patients in their early childhood.…”

Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells

“…The relationship between CFTR and the altered mucus phenotype in CF may be explained by intracellular effects and/or extracellular processes (26). Support for the former is provided by reports that mucin gene expression (15,29) and glycosylation profiles (9,42,45) of mucins from CF patients or rodent models of CF are altered.…”

“…Evidence in support of the latter is provided by reports demonstrating that features of the extracellular milieu such as ionic concentration and pH affect the rheology of the secreted mucous gel (10,30,33). Secondary effects due to infection, inflammation, or drugs further cloud the relationship (27), as does the use of in vitro systems of neoplastic origin or expressing nonnative mucins (26). Conversely, several studies have shown no direct relationship between CFTR expression and mucin characteristics such as sulfation (7,18,26).…”

CFTR, Mucins, and Mucus Obstruction in Cystic Fibrosis

“…With the identification of CFTR and the suggestion that its effective absence from organelle and plasma membranes in CF cells was responsible for alkalization and dysfunction along the secretory pathway (Barasch et al 1991;Barasch and alAwqati 1993), a possible mechanism was suggested. After many years of controversy, which led to increasingly sophisticated studies, the issue appears to have been resolved with the weight of the evidence indicating the physical absence of CFTR in the Golgi apparatus (see above), and no apparent role for CFTR in the maintenance of Golgi pH (Seksek et al 1996;Gibson et al 2000;Chandy et al 2001;Haggie and Verkman 2009) or in the glycosylation of mucins and other glycoproteins (Jiang et al 1997;Holmen et al 2004;Leir et al 2005). Biochemical analyses of O-glycans indicate that changes in glycosylation of CF mucins appear to be a secondary effect of the disease that is largely due to inflammation (Davril et al 1999;Roussel and Lamblin 2003;Schulz et al 2007).…”