ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis
Nanette Kälin, Andreas Claaß, Martin Sommer, Edith Puchelle, Burkhard Tümmler
Nanette Kälin, Andreas Claaß, Martin Sommer, Edith Puchelle, Burkhard Tümmler
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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

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CFTR expression in the intestine. (a) CFTR immunoreactive signals of the...
CFTR expression in the intestine. (a) CFTR immunoreactive signals of the intestine are absent in an internal negative control, a tissue specimen homozygous for 2 null alleles. CFTR immunoanalysis of a rectal biopsy from a patient homozygous for an out-of-frame deletion of exon 2 and 3 (2a and 2b) in comparison with non-CF (1a and 1b) and ΔF508 homozygous CF patients (3a and 3b), using CFTR antibody MATG1104 (1:200). Fluorescence exposure times for 1b, 2b, and 3b were 30 sec. “a” panels show Nomarski microscopy of indirect CFTR immunofluorescence in “b” panels. ×1,241. (b) General view of CFTR labeling patterns in duodenum, jejunum, and ileum of adult non-CF intestine (1–3) and newborn ileum from a CF patient suffering from meconium ileus (4b) with CFTR antibody PAC865 (1:200). Fluorescence exposure times for duodenum: 15 sec; jejunum: 30 sec; adult non-CF ileum: 30 sec; newborn ΔF508 CF ileum: 30 sec. “a” panels show Nomarski microscopy of indirect CFTR immunofluorescence in “b” panels. ×616. (c) CFTR immunolabeling of the intestine was compared with the signal pattern of marker proteins of different cellular membranes. CFTR expression in isolated intestinal cells of the villi in duodenum and jejunum (2a and 2b, arrow; MATG1104, 1:200) appears apical when compared with a marker of the luminal cell membrane of intestinal villi (1a and 1b; alkaline phosphatase). Similar immunoreactive labeling patterns of CFTR (4a and 4b; PAC13, 1:200) and a marker protein for the ER (3a and 3b; p63) in intermediate crypts of jejunum, labeling of both proteins predominantly within mucus-secreting cells. Identical CFTR immunoreactivity in deep crypts of the ileum of non-CF (5a and 5b; PAC865, 1:200) and ΔF508 homozygous CF patients (6a and 6b; PAC865, 1:200). Fluorescence exposure times: 1b = 20 sec; 2b = 60 sec; 3b = 60 sec; 4b = 30 sec; 5b = 15 sec; 6b = 15 sec. “a” panels show Nomarski microscopy of indirect CFTR immunofluorescence in “b” panels. ×2,467. (d) CFTR immunolabeling in paraffin-embedded intestinal tissue specimens with PAC13 (1:50) and signal detection with horseradish peroxidase and DAB/nickel chloride as chromogen substrate. CFTR immunolabeling within intestinal goblet cells (1a) was forced out by peptide competition with CFTR immunogen (CFTR 1468–1480) (1b). Comparison of CFTR immunolabeling of non-CF (2a) and ΔF508 homozygous CF ileum (2b) in general view. CFTR immunolocalization within goblet cells in a high-magnification view demonstrates that CFTR signals are intracellular (3b) and overlap with the spatial distribution of the trans-Golgi network, as indicated by γ-adaptin immunolabeling (3a). CFTR labeling within goblet cells is indistinguishable in subcellular localization and intensity in non-CF (4a and 5a) and ΔF508 homozygous CF tissues (4b and 5b). 1a, 1b, 2a, 2b: ×616. 3a, 3b, 4a, 4b, 5a, 5b: ×6,168.