Moll R, Schiller D L, Franke W W
Institute of Pathology, University of Mainz Medical School, Federal Republic of Germany.
J Cell Biol. 1990 Aug;111(2):567-80. doi: 10.1083/jcb.111.2.567.
A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.
采用生化和免疫学方法对人肠上皮细胞的一种主要细胞骨架多肽（分子量约46,000；蛋白IT）进行了表征。该多肽经体外翻译被鉴定为一种特异性的真实mRNA产物，在SDS - PAGE后的免疫印迹中，仅与所检测的众多细胞角蛋白（CK）抗血清中的一种发生反应，而与许多单克隆CK抗体均无反应。体外实验表明，它与II型CK 8形成异型复合物，这通过印迹结合试验和在4M尿素中的凝胶电泳得以证实，并且这些复合物在适当条件下组装成中间丝（IFs）。从细胞骨架IFs中可获得一种对胰凝乳蛋白酶有抗性的、分子量约38,000的蛋白IT核心片段，这表明它包含在卷曲螺旋结构中。针对蛋白IT产生的抗体可识别正常和转化的肠细胞中典型的CK纤维阵列。从纯化的多肽IT获得的四个蛋白水解肽片段与其他几种I型CK杆状结构域的螺旋I和螺旋II的相应区域表现出显著的氨基酸序列同源性。免疫细胞化学检测显示，该蛋白在肠和胃小凹上皮、尿路上皮伞细胞以及表皮的默克尔细胞中特异性表达，呈显著成分。在胸腺、支气管、胆囊和前列腺中可发现少量阳性上皮细胞。蛋白IT的表达在原发性和转移性结直肠癌以及由此衍生的细胞培养物中通常得以维持。在其他几种哺乳动物物种中也发现了相应的蛋白。我们得出结论，多肽IT是一种完整的IF成分，尽管与I型CKs的关系较为疏远，但仍与之相关，因此，我们建议将其作为CK 20添加到人类CK目录中。
