title: 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 (DeltaF508 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.
authors.affiliation: Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
authors.forename: Shih-Hsing
authors.initials: SH
authors.lastname: Leir
authors.forename: Simon
authors.initials: S
authors.lastname: Parry
authors.forename: Timea
authors.initials: T
authors.lastname: Palmai-Pallag
authors.forename: Joanne
authors.initials: J
authors.lastname: Evans
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Ann
authors.initials: A
authors.lastname: Harris
mesh_terms: D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D015394:Molecular Structure; D018396:Mucin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013431:Sulfates / Q000378:metabolism*
chemical_list: C505032:CFTR protein, human; D018396:Mucin-1; D011134:Polysaccharides; D013431:Sulfates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator