ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis
Nanette Kälin, … , Edith Puchelle, Burkhard Tümmler
Nanette Kälin, … , Edith Puchelle, Burkhard Tümmler
Published May 15, 1999
Citation Information: J Clin Invest. 1999;103(10):1379-1389. https://doi.org/10.1172/JCI5731.
View: Text | PDF
Article

ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis

Abstract

Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation ΔF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from ΔF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In ΔF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas ΔF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of ΔF508 CFTR expression from null to apparently normal amounts indicates that ΔF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in ΔF508 CF respiratory and intestinal disease.

Authors

Nanette Kälin, Andreas Claaß, Martin Sommer, Edith Puchelle, Burkhard Tümmler

×

Figure 5

Options: View larger image (or click on image) Download as PowerPoint
Immunoblot analysis of CFTR expression in nasal polyps and the intestine...
Immunoblot analysis of CFTR expression in nasal polyps and the intestine of non-CF and ΔF508 homozygous CF patients. Discrimination of CFTR glycoforms by deglycosylation (generation of unglycosylated band A) with N-glycosidase F and endoglycosidase H; the latter reacts with core-glycosylated CFTR (band B) but not with mature CFTR (band C). Deglycosylations were for 3 hours at 37°C in the presence of protease inhibitors. (a) The major CFTR immunoreactive bands of non-CF and ΔF508 homozygous CF nasal polyps and intestinal tissue specimens were indistinguishable from complex-glycosylated band C of T84 cells. In the intestine, CFTR expression was highest in membrane preparations from duodenum and decreased in tissue specimens from jejunum and ileum. Immunodetection was performed with MATG1104 (1:500). (b) Complex modification of the major CFTR immunoreactive signal of ΔF508 homozygous CF intestine (right) is demonstrated by sensitivity to N-glycosidase F and resistance to endoglycosidase H, as is the case for mature CFTR of control non-CF intestine (middle) and T84 cells (left). Immunodetection with M3A7 (1:500). (c) Specificity of the immunoreactive bands from intestinal tissue specimens for CFTR is demonstrated by the characteristic mobility shifts in deglycosylation assays with N-glycosidase F and endoglycosidase H and by peptide competition for antibody binding with the CFTR immunogen. (1) From adult non-CF duodenum; (2) from jejunum of the same patient; (3) from adult ileum of a ΔF508 homozygous CF patient. “a” lanes show deglycosylation of the membrane preparations with N-glycosidase F. “b” lanes show peptide competition with the CFTR immunogen (1 μg MATG1104 preincubated at 20°C with 5 μg CFTR peptide 722–734). Immunodetection with MATG1104 (1:500).