Sarkadi B, Bauzon D, Huckle W R, Earp H S, Berry A, Suchindran H, Price E M, Olson J C, Boucher R C, Scarborough G A
Department of Medicine, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1992 Jan 25;267(3):2087-95.
Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.
针对两个人类囊性纤维化跨膜传导调节因子（CFTR）的R结构域和C末端对应的合成肽产生的亲和纯化多克隆抗体，用于在人类上皮细胞中对该蛋白质进行表征和定位。采用一种能有效检测大型疏水蛋白质的免疫印迹技术，两种抗体均能识别正常和囊性纤维化（CF）患者气道上皮细胞以及T84结肠癌细胞的细胞裂解物和分离膜中的一种约180 kDa的蛋白质。通过有限的羧肽酶Y消化消除了与抗C末端抗体的反应性，但未消除与抗R结构域抗体的反应性。当正常CFTR cDNA通过逆转录病毒载体在CF或正常气道上皮细胞或小鼠成纤维细胞中过表达时，产生的蛋白质表观分子量约为180 kDa。通过杆状病毒载体在昆虫（Sf9）细胞中表达的CFTR分子量约为140 kDa，可能代表一种非糖基化形式。上皮细胞中的CFTR似乎以几种形式存在。对T84细胞膜进行N-糖苷酶处理可使主要CFTR条带的表观分子量从180 kDa降至140 kDa，但T84细胞CFTR的一部分无法去糖基化，气道上皮细胞膜中的CFTR也无法去糖基化。此外，麦胚凝集素可从去污剂溶解的T84细胞膜中吸收大部分CFTR，但不能从气道细胞膜中吸收。发现所有上皮细胞类型中的CFTR都是一种整合膜蛋白，不会因高盐或二碘水杨酸锂处理而溶解。对先前通过酶促半乳糖苷化进行表面标记的气道上皮细胞制备的粗膜进行蔗糖密度梯度分级分离，结果显示正常CFTR和携带Phe508缺失（ΔF 508）的CFTR均定位于质膜。在所有情况下，CFTR均与Na +、K（+）-ATP酶和质膜钙ATP酶共定位，而内质网钙ATP酶和线粒体膜标记物在较高蔗糖密度下富集。因此，CFTR似乎在正常和ΔF 508 CF上皮细胞中均定位于质膜。
