Dataset Information

Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene.

ABSTRACT: There is a need for a large-animal model to investigate the etiology and biology of cystic fibrosis (CF) lung disease and to study potential therapies. The development and electrophysiology of the sheep airway have been shown to exhibit close functional parallels with the human airway, particularly with respect to the respiratory epithelium. We have cloned and sequenced the ovine cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. It shows a high degree of conservation at the DNA coding and predicted polypeptide levels with human CFTR: at the nucleic acid level there is a 90% conservation (compared with 80% between human and mouse CFTR cDNA); at the polypeptide level, the degree of similarity is 95% (compared with 88% between human and mouse). Northern blot analysis and reverse transcription-PCR have shown that the patterns of expression of the ovine CFTR gene are very similar to those seen in humans. Further, the developmental expression of CFTR in the sheep is equivalent to that observed in humans. Thus, overall a CF sheep should show lung pathology similar to that of humans with CF.

SUBMITTER: Tebbutt SJ

PROVIDER: S-EPMC42470 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene.

Tebbutt S J SJ Wardle C J CJ Hill D F DF Harris A A

Proceedings of the National Academy of Sciences of the United States of America 19950301 6

There is a need for a large-animal model to investigate the etiology and biology of cystic fibrosis (CF) lung disease and to study potential therapies. The development and electrophysiology of the sheep airway have been shown to exhibit close functional parallels with the human airway, particularly with respect to the respiratory epithelium. We have cloned and sequenced the ovine cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. It shows a high degree of conservation at the DNA co ...[more]

PMID: 7534416

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene.

Publications

Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene.

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