title: Cystic fibrosis mutations lead to carboxyl-terminal fragments that highlight an early biogenesis step of the cystic fibrosis transmembrane conductance regulator.
abstract: Inefficient delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to the surface of cells contributes to disease in the majority of cystic fibrosis patients. Analysis of cystic fibrosis-associated missense mutations in the first nucleotide binding domain (NBD1), including A455E, S549R, Y563N, and P574H, revealed reduced levels of mature CFTR with elevated levels of carboxyl-terminal polypeptide fragments of 105 and 90 kDa. These fragments appear early in biogenesis and degrade rapidly in four distinct cell types tested including the bronchial epithelial IB3-1 cell line. They were detected at highest levels with CFTRA455E where the 105-kDa fragment accounted for 40% of newly synthesized polypeptide but for only 20 and 7% of nascent wild type and mutant DeltaF508 proteins, respectively. The bands represent core- and unglycosylated forms of the same CFTR fragment supporting that precursor forms are correctly inserted into the membrane of the endoplasmic reticulum. Proteolytic cleavage would be predicted to occur on the cytosolic face of the endoplasmic reticulum within the NBD1-R domain segment, but pharmacological testing did not support involvement of the 26 S proteasome. The examined missense mutations in NBD1 manifest differently than the major mutant, DeltaF508, and highlight a critical conformational aspect of biogenesis of CFTR.
authors.affiliation: Department of Molecular and Medical Genetics, Program in Genetics & Genomic Biology, University of Toronto, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Van Oene
authors.forename: G L
authors.initials: GL
authors.lastname: Lukacs
authors.forename: J M
authors.initials: JM
authors.lastname: Rommens
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D020125:Mutation, Missense; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D000939:Epitopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator