Mall Marcus, Kreda Silvia M, Mengos April, Jensen Timothy J, Hirtz Stephanie, Seydewitz Hans H, Yankaskas James, Kunzelmann Karl, Riordan John R, Boucher Richard C
School of Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.
Gastroenterology. 2004 Jan;126(1):32-41. doi: 10.1053/j.gastro.2003.10.049.
Deletion of the codon for phenylalanine at position 508 (DeltaF508) is the most frequent disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In heterologous cells, defective processing of the DeltaF508 protein results in endoplasmic reticulum retention, proteolytic degradation, and absence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent plasma membrane Cl(-) conductance. However, data with respect to the processing block of DeltaF508 protein in native epithelia are limited and conflicting.
To characterize both the fate and function of DeltaF508 protein in a native epithelium, we measured CFTR-mediated Cl(-) secretion, localization of the CFTR protein, and CFTR maturation in rectal biopsy specimens from normal individuals and DeltaF508 homozygous patients with cystic fibrosis (CF).
Ussing chamber studies showed that cAMP-dependent and cholinergic Cl(-) secretion was absent from rectal tissues freshly excised from DeltaF508 homozygous patients with CF. By immunohistochemistry, we detected wild-type but not DeltaF508 CFTR at the luminal membrane of crypt colonocytes. By sequential immunoprecipitation and immunoblotting analyses, mature CFTR protein was detected in normal but not in DeltaF508 homozygous tissues.
Collectively, these data show that there is insufficient maturation and transport of DeltaF508 CFTR from the endoplasmic reticulum to the apical membrane to support CFTR-mediated Cl(-) secretion in the CF colon.
囊性纤维化跨膜传导调节因子（CFTR）基因中第508位苯丙氨酸密码子缺失（ΔF508）是最常见的致病突变。在异源细胞中，ΔF508蛋白的加工缺陷导致其在内质网中滞留、蛋白水解降解，且缺乏依赖于3',5'-环磷酸腺苷（cAMP）的质膜氯离子（Cl⁻）传导。然而，关于天然上皮细胞中ΔF508蛋白加工障碍的数据有限且相互矛盾。
为了表征天然上皮细胞中ΔF508蛋白的命运和功能，我们检测了正常个体以及患有囊性纤维化（CF）的ΔF508纯合患者直肠活检标本中CFTR介导的Cl⁻分泌、CFTR蛋白的定位以及CFTR的成熟情况。
尤斯灌流小室研究表明，从患有CF的ΔF508纯合患者新鲜切除的直肠组织中不存在依赖于cAMP和胆碱能的Cl⁻分泌。通过免疫组织化学，我们在隐窝结肠细胞的腔膜上检测到了野生型CFTR，但未检测到ΔF508 CFTR。通过连续免疫沉淀和免疫印迹分析，在正常组织中检测到了成熟的CFTR蛋白，而在ΔF508纯合组织中未检测到。
总体而言，这些数据表明，在CF结肠中，ΔF508 CFTR从内质网到顶端膜的成熟和转运不足以致于无法支持CFTR介导的Cl⁻分泌。
