IDENTIFICATION OF A 160-KDA POLYPEPTIDE THAT BINDS TO THE TIGHT JUNCTION PROTEIN-ZO-1
IDENTIFICATION OF A 160-KDA POLYPEPTIDE THAT BINDS TO THE TIGHT JUNCTION PROTEIN-ZO-1
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DOI：
10.1073/pnas.88.8.3460
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发表时间：
1991-04-01
影响因子：
11.1
通讯作者：
APATIRA, D
中科院分区：
文献类型：
--
作者：
GUMBINER, B;LOWENKOPF, T;APATIRA, D
ZO-1是一种210- 220-kDa的外周膜蛋白，与上皮紧密连接的细胞质表面相关。 由于ZO-1可能与其他未识别的紧密连接蛋白相互作用，我们已经寻找了其他多肽结合ZO-1。 一个160 kDa的多肽被确定为与ZO-1从代谢标记的Madin-Darby犬肾（MDCK）细胞的洗涤剂提取物共免疫沉淀。 根据几个标准，该多肽似乎与ZO-1不同，而不是降解产物。 它缺乏ZO-1单克隆抗体和多克隆血清识别的ZO-1表位，因为它在全细胞提取物或ZO-1免疫沉淀物的免疫印迹中检测不到。 此外，在一维“Cleveland凝胶”上，它显示出与ZO-1不同的肽图谱。“此外，由于新合成的160 kDa多肽在抗ZO-1免疫沉淀物中出现的动力学比ZO-1慢得多，因此它在免疫沉淀物中的存在不能简单地用细胞裂解过程中ZO-1的降解来解释。 像ZO-1一样，160-kDa多肽似乎是细胞质外周膜蛋白。 它不能被两种不同的细胞表面标记试剂标记。 它可以在没有洗涤剂的情况下通过高盐浓度从膜中提取。 正如蛋白质复合物所预期的，160-kDa多肽和ZO-1以相似的动力学翻转。 我们建议，160 kDa的多肽是一个组成部分的紧密连接。
ZO-1 is a 210- to 220-kDa peripheral membrane protein associated with the cytoplasmic surface of the epithelial tight junction. Because ZO-1 may interact with other unidentified tight junction proteins, we have looked for other polypeptides that bind to ZO-1. A 160-kDa polypeptide was identified that coimmunoprecipitates with ZO-1 from detergent extracts of metabolically labeled Madin-Darby canine kidney (MDCK) cells. This polypeptide appears to be distinct from ZO-1, rather than a degradation product, by several criteria. It lacks ZO-1 epitopes recognized by both monoclonal antibodies and a polyclonal serum to ZO-1, since it is not detectable in immunoblotsof either whole cell extracts or ZO-1 immunoprecipitates. Also, it exhibits a peptide map different from that of ZO-1 on one-dimensional "Cleveland gels." Moreover, because the kinetics of appearance of newly synthesized 160-kDa polypeptide in anti-ZO-1 immunoprecipitates is much slower than that of ZO-1, its presence in immunoprecipitates cannot be simply explained by degradation of ZO-1 during cell lysis. Like ZO-1, the 160-kDa polypeptide seems to be a cytoplasmic peripheral membrane protein. It cannot be labeled by two different cell surface labeling reagents. It can be extracted from the membrane by high salt concentration in the absence of detergents. As expected for protein complex, the 160-kDa polypeptide and ZO-1 turn over with similar kinetics. We propose that the 160-kDa polypeptide is a component of the tight junction.