Volume 126, Issue 1, Pages 32-41 (January 2004) The ΔF508 mutation results in loss of CFTR function and mature protein in native human colon  Marcus Mall, Silvia M. Kreda, April Mengos, Timothy J. Jensen, Stephanie Hirtz, Hans H. Seydewitz, James Yankaskas, Karl Kunzelmann, John R. Riordan, Richard C. Boucher  Gastroenterology  Volume 126, Issue 1, Pages 32-41 (January 2004) DOI: 10.1053/j.gastro.2003.10.049

Figure 1 Cholinergic and cAMP-dependent activation of ion transport in native normal and CF rectal epithelia. Experiments were performed in the presence of indomethacin (10 μmol/L, basolateral) and amiloride (10 μmol/L, luminal). Original recordings of the effects of carbachol (cch) and IBMX/forskolin on Vte and Rte in (A) normal and (B) CF (ΔF508/ΔF508) rectal biopsy specimens. Rte was determined from Vte deflections obtained by pulsed current injection. Time gaps between recordings were 20 minutes. (C) Summary of cAMP-induced (IBMX/forskolin) Isc in rectal biopsy specimens from wild-type (wt/wt), ΔF508 heterozygous (ΔF/wt), and ΔF508 homozygous CF (ΔF/ΔF) subjects. Individual data points represent the mean of measurements on 2–5 biopsy specimens per individual. ∗Significant difference comparing the effects of IBMX/forskolin in normal (wt/wt or ΔF/wt) and CF (ΔF/ΔF) rectal tissues (Kruskal-Wallis analysis of variance on ranks). (D) Summary of carbachol-induced Isc in the absence (−) and presence (+) of IBMX/forskolin. Closed circles, initial peak responses; open circles, the plateau for monophasic responses or the lumen-negative peak for biphasic responses in the absence of cAMP-dependent activation; closed diamonds, initial peak responses; open diamonds, the plateau for monophasic lumen-positive responses or the lumen-negative peak for biphasic responses in the presence of cAMP-dependent activation. #Significantly different from carbachol-induced plateau responses in rectal tissues from wild-type (wt/wt) and ΔF508 heterozygous (ΔF/wt) subjects in the absence of cAMP-dependent stimulation. ∗Significantly different from peak responses in wt/wt and ΔF/wt subjects in the presence of cAMP-dependent stimulation (Kruskal-Wallis analysis of variance on ranks). Solid line, median; dashed line, 25th and 75th percentiles, respectively. Gastroenterology 2004 126, 32-41DOI: (10.1053/j.gastro.2003.10.049)

Figure 2 CFTR protein detection in heterologous cells. (A) Specificity and sensitivity of monoclonal CFTR antibodies. (A, left panel) MTE18 cells lacking (m) or expressing (c) human CFTR were assayed by Western blot with identical concentrations (1 μg/mL) of monoclonal CFTR antibodies 769, 528, 596, and 217 (new) and 24-1 and MATG1061 (published). Mature (arrow) and immature (arrowhead) CFTR bands are indicated. (A, right panel) MTE18 cells lacking (mock) or expressing human CFTR grown on collagen-coated glass culture chambers were assayed independently for immunofluorescence with the indicated CFTR antibodies at identical concentrations (1 μg/mL). Nuclei were stained with 4′,6-diamidino-2-phenylindole. Cells were analyzed with a Leica 4D-TCS confocal microscope by xz scanning using 2 independent laser sources. (Original magnification 150×.) (B) BHK cells lacking (mock) or expressing either wild-type (wt) or ΔF508 (ΔF) CFTR were assayed by Western blot and immunofluorescence as previously described using monoclonal CFTR antibodies 769, 528, 596, and 217 as indicated. The mature CFTR band (arrow) is only present in BHK wt CFTR cells. The doublet evident in the ΔF508 BHK cells reflects the full-length core glycosylated protein and an N-terminally cleaved proteolysis product (upper and lower band, respectively). Molecular-weight bands are indicated in kilodaltons. Gastroenterology 2004 126, 32-41DOI: (10.1053/j.gastro.2003.10.049)

Figure 3 CFTR protein detection in freshly excised human colonic epithelia. Rectal biopsy specimens from normal (wt/wt; n = 3), ΔF508 homozygous (ΔF/ΔF; n = 3), and ΔF508 heterozygous (ΔF/wt; n = 3) individuals and the ascending colon of a normal individual (∗) were homogenized and immunoprecipitated with a polyclonal CFTR antibody. After sodium dodecyl sulfate/polyacrylamide gel electrophoresis (6% acrylamide), immunoblots were probed with a cocktail of CFTR monoclonal antibodies 596 and 217, which recognize different CFTR epitopes (see Patients and Methods). The fully glycosylated CFTR protein (arrow) was only observed in normal specimens. In contrast, CF samples exhibited only the immature CFTR protein (arrowhead). ΔF508 heterozygous specimens exhibited both fully processed and immature protein forms. Specimens from the rectum and ascending colon showed a similar pattern of CFTR protein expression. Molecular-weight bands are indicated in kilodaltons. Gastroenterology 2004 126, 32-41DOI: (10.1053/j.gastro.2003.10.049)

Figure 4 Morphology of freshly excised human rectal epithelia. (A) Schematic of rectal epithelium showing a transverse section of a deep crypt (left) and a single crypt (center). Colonocytes are shown in green and goblet cells in purple; nuclei are represented in blue. The actin cytoskeleton was labeled with Alexa 488 phalloidin to identify colonocytes and MUC2 antibody (color encode in purple) was used to label goblet cells (far right). l, lumen. (B) Rectal biopsy specimens from 10 normal (wt/wt) and 8 ΔF508 homozygous patients with CF (ΔF/ΔF) were immunostained with a MUC2 monoclonal antibody followed by a Texas Red-labeled secondary antibody and Alexa 488 phalloidin. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Images show xy confocal planes scanned in a Leica SP2 AOBS confocal microscope in the differential interference contrast, 4′,6-diamidino-2-phenylindole, Texas Red (color encode in purple), and Alexa 488 channels using 3 independent laser sources. The antibody recognizes the MUC2 precursor protein and stains mainly the MUC2 associated with the endoplasmic reticulum of the goblet cells in both normal and CF crypts. (Bar = 20 μm.) Gastroenterology 2004 126, 32-41DOI: (10.1053/j.gastro.2003.10.049)

Figure 5 CFTR immunolocalization in freshly excised human rectal epithelia. Rectal biopsy specimens from 10 normal (wt/wt) and 8 age-matched ΔF508 homozygous individuals with CF (ΔF/ΔF) were immunostained independently with 3 CFTR antibodies (528, 769, and 525) and control immunoglobulin G at identical concentrations (1 μg/mL) followed by a Texas Red-labeled secondary antibody and Alexa 488 phalloidin (F-actin). Images are xy confocal planes recorded in the differential interference contrast, Texas Red, and Alexa 488 channels using 2 independent laser sources (488 and 568 nm) and identical scanning conditions. Representative results from (A) 1 normal and (B) 1 CF rectal tissue, stained with antibody 528, are shown. Control immunoglobulin G staining was negative in both normal and CF epithelia. Arrowheads indicate the position of the apical membrane of colonocytes. (Original magnification 100×.) Gastroenterology 2004 126, 32-41DOI: (10.1053/j.gastro.2003.10.049)