A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models

Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires.     

The porcine lung as a potential model for cystic fibrosis

Airway disease currently causes most of the morbidity and mortality in patients with cystic fibrosis (CF). However, understanding the pathogenesis of CF lung disease and developing novel therapeutic strategies have been hampered by the limitations of current models. Although the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) has been targeted in mice, CF mice fail to develop lung or pancreatic disease like that in humans. In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. Here, we review aspects of porcine airways and lung that are relevant to CF.     

Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.

The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.     

Characterization of the oligosaccharide structures associated with the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane-associated glycoprotein. The protein can exist in three different molecular weight forms of approximately 127, 131, and 160 kDa, representing either nonglycosylated, core glycosylated, or fully mature, complex glycosylated CFTR, respectively. The most common mutation in cystic fibrosis (CF) results in the synthesis of a variant (DeltaF508-CFTR) that is incompletely glycosylated and defective in its trafficking to the cell surface. In this study, we have analyzed the oligosaccharide structures associated with the different forms of recombinant CFTR, by expressing and purifying the channel protein from either mammalian Chinese hamster ovary (CHO) or insect Sf9 cells. Using glycosidases and FACE analysis (fluorophore-assisted carbohydrate electrophoresis) we determined that purified CHO-CFTR contained polylactosaminoglycan (PL) sequences, while Sf9-CFTR had only oligomannosidic saccharides with fucosylation on the innermost GlcNAc. The presence of PL sequences on the recombinant CHO-CFTR is consistent with a normal feature of mammalian processing, since endogenous CFTR isolated from T84 cells displayed a similar pattern of glycosylation. The present study also reports on the use of FACE for the qualitative analysis of small amounts of glycoprotein oligosaccharides released enzymatically.     

XV-2c/KM19 haplotypes analysis of cystic fibrosis patients from western Mexico

The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects. The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background. Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.     

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (～10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.     

Gene therapy for cystic fibrosis

Cystic fibrosis (CF) is associated with significant morbidity and mortality, despite significant advances in conventional treatment. The field of gene therapy has progressed rapidly since the cystic fibrosis transmembrane conductance regulator (CFTR) gene was cloned. In this review we discuss current knowledge on the underlying molecular defect in CF, and the progress in gene transfer studies from the early in vitro work through to clinical trials, including the development of endpoints to assess efficacy. We highlight the problems encountered, and likely future directions of the field.     

The basic defect in cystic fibrosis.

Recent evidence strongly suggests that the cystic fibrosis gene product (CFTR) is a Cl- channel. Its properties, however, differ from those of a 30-50 pS outwardly rectifying channel previously implicated as defective in cystic fibrosis. It is still uncertain whether the pleiotropic effects of the CF defect, such as increased airway Na+ absorption and mucus sulfation, are secondary to reduced Cl- conductance, or reflect additional functions of CFTR.     

Infrared biomarkers of impaired cystic fibrosis transmembrane regulator protein biogenesis

The mid‐infrared (IR) spectra of human cystic fibrosis (CF) cells acquired by Fourier transform infrared microspectroscopy were compared with those of non‐CF cells. Within the 1700 to 1480 cm^?1 spectral domain of amides, unsupervised explorative principal component analysis identified a few variables reflecting quantitative and qualitative vibrations arising from protein secondary structures and amino acid side chains. Their pattern reflected α‐helix to β‐sheet transitions in bronchial epithelial cells and in immortalized peripheral blood mononuclear cells from patients with R1162X missense or in‐frame F508del mutations in the cystic fibrosis transmembrane regulator gene (Cftr). Similar transitions have been described in IR spectra of cells, tissues and body fluids of patients affected with some neurodegenerative diseases characterized by the accumulation of misfolded protein aggregates. The variables pattern was able to distinguish CF cells from non‐CF cells and was modified by molecular compounds used to rescue the unbalanced folding process of mutated cystic fibrosis transmembrane regulator (CFTR) anion channel. To our knowledge, this is the first experimental evidence of spectroscopic biomarkers of the impaired biogenesis of CFTR by IR microanalysis in the spectra of human CF bronchial epithelial and lymphoblastoid cells.     

Applicability of different antibodies for immunohistochemical localization of CFTR in sweat glands from healthy controls and from patients with cystic fibrosis.

The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, DeltaF508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only three-M3A7, MATG 1104, and cc24-met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of DeltaF508 CFTR. The antibodies exhibited different sensitivities for detecting DeltaF508 CFTR.     

Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and cGMP-regulated chloride channel critical to the regulation of intestinal fluid, chloride, and bicarbonate secretion. In cystic fibrosis (CF), mutations in CFTR result in downregulation of CFTR function and small intestinal obstruction. Unlike the human CF intestine, severe gastrointestinal disease and lethal obstruction is common in transgenic mice deficient in CFTR. The relevance of the physiology of CFTR and pathophysiology of CF in genetically altered mice to that of human CF disease remains incompletely understood. We hypothesized that the expression and distribution of CFTR in mouse intestine may differ from that of human and may contribute to the variation in disease expression between the two species. Using immunocytochemical and immunoblot techniques and well-characterized anti-rodent anti-CFTR antibodies, we examined the cellular distribution of CFTR in the mouse intestinal tract. We identified significant differences in villus distribution for CFTR in the mouse proximal small intestine compared to those previously reported for human and rat. These observations are important to the understanding of CFTR pathophysiology in transgenic CF mouse model systems and bear relevance to the different phenotypic expression of disease in mice compared to human.     

Increased expression of cystic fibrosis transmembrane conductance regulator in rat liver after common bile duct ligation

Liver disease associated with cystic fibrosis (CF) has been increasingly diagnosed and recognized as one of the major causes of death in CF during recent years. The autosomal-recessive disorder of CF results from mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) that encodes the CFTR protein. Due to its existence and multifunction in biliary epithelial, over- or less-expression of CFTR in the liver may play an important role in the development of CF liver disease (CFLD). The aim of current study is to investigate the expression of CFTR in the liver of common bile duct ligated (BDL) rats. After BDL, there was an increase in the abundance of CFTR mRNA and protein. Immunohistochemical staining also demonstrated an increased intensity of CFTR staining in the liver tissue section. In conclusion, there is an increased expression of CFTR in the liver after common BDL.     

Bacterial host interactions in cystic fibrosis

Chronic infection is a hallmark of cystic fibrosis (CF) and the main contributor to morbidity. Microbial infection in CF is complex, due to the number of different species that colonise the CF lung. Their colonisation is facilitated by a host response that is impaired or compromised by highly viscous mucous, zones of hypoxia and the lack of the cystic fibrosis transmembrane regulator (CFTR). Successful dominant CF pathogens combine an effective arsenal to establish infection and counter-attack the host response, together with an ability to adapt readily to an unfavourable environment. Hypermutability is common among CF pathogens facilitating adaptation and as the host response persists, progressive destruction of the normal architecture of lung tissue ensues with catastrophic consequences for the host.     

Glycosylation and the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.     

Interest of colchicine for the treatment of cystic fibrosis patients. Preliminary report

Cystic fibrosis (CF) lung disease is characterized by persistent inflammation. Antiinflammatory drugs, such as corticosteroids and ibuprofen, have proved to slow the decline of pulmonary function although their use is limited because of frequent adverse events. We hypothesized that colchicine could be an alternative treatment because of its antiinflammatory properties and upregulatory effect on cystic fibrosis transmembrane regulator (CFTR) closely related proteins. We herein present results obtained in an open study of eight CF children treated with colchicine for at least 6 months. Clinical status was better in all patients and respiratory function tests significantly improved in five. Median duration of antibiotherapy decreased significantly. These preliminary results support our hypothesis of a beneficial effect of colchicine in CF patients and stress the need for a controlled therapeutic trial.     

Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.     

Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia.     

Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity

Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels.