Riopel C L, Butt I, Omary M B
Department of Medicine, Palo Alto VA Medical Center, California 94304.
Cell Motil Cytoskeleton. 1993;26(1):77-87. doi: 10.1002/cm.970260108.
Keratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well-accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase-catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water-soluble sulfo-NHS-biotin. Partitioning of the keratins to a soluble and an insoluble pool after "cell surface" 125I-labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti-keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques.
角蛋白是大多数上皮细胞中发现的细胞骨架中间丝蛋白的一个亚群。一些报告表明，角蛋白可能在细胞表面被发现，以及它们被广泛认可的细胞骨架位置。解释角蛋白细胞表面表达的主要证据是细胞表面放射性碘化。在这里，我们表明，当对结肠和乳腺组织培养细胞进行处理时，乳过氧化物酶催化的碘化作用会导致角蛋白被放射性标记。当细胞直接在组织培养皿上标记时，未检测到角蛋白的标记。当使用水溶性磺基-NHS-生物素对完整细胞进行生物素化时，也获得了类似的结果。在“细胞表面”125I标记后，角蛋白分为可溶性和不溶性部分，结果表明这两个部分都被碘化。间接免疫荧光显示，一组抗角蛋白抗体与完整上皮细胞的结合仅发生在更贴壁的细胞上，即需要更长时间处理才能从组织培养皿中移除的细胞。综上所述，我们的结果表明，在某些细胞中报道的细胞表面角蛋白表达可能反映的是细胞内角蛋白。此外，上皮细胞处理方法可以显著改变细胞表面碘化技术的渗漏情况。
