Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein.
Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein.
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DOI：
10.1083/jcb.124.6.949
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发表时间：
1994-03
期刊：
影响因子：
--
通讯作者：
Goodenough DA
中科院分区：
文献类型：
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作者：
Jesaitis LA;Goodenough DA
ZO-1是一种210-225 kD的外周膜蛋白，与闭锁带或紧密连接的细胞质表面相关。在Gumbiner，B.，T.Lowenkopf和D.Apatira的条件下制备的MDCK细胞提取液中，发现一种160kD的多肽，命名为ZO-2，与ZO-1免疫共沉淀。1991年。程序娜塔莉。阿卡德。SCI。美国。88：3460-3464)。我们用ZO-1与MDCK细胞共沉淀，然后从制备的SDS-PAGE凝胶切片上电洗脱，从MDCK细胞单层中分离出ZO-2。利用从ZO-2胰酶片段获得的氨基酸序列信息，从MDCK文库中分离出部分cDNA克隆。推导的氨基酸序列表明，犬ZO-2含有一个与人和小鼠ZO-1序列非常相似的区域。该区域包括一个功能未知的90个氨基酸重复结构域和鸟苷酸激酶样结构域，这些结构域在蛋白质家族成员中共享，其中包括ZO-1，红细胞P55，果蝇致死(1)Diss-Large-1(DLG)基因的产物，以及大鼠脑中的突触相关蛋白PSD-95/SAP90。DLG基因产物已被证明在果蝇幼虫的成像盘中发挥肿瘤抑制作用，尽管其他家族成员的功能尚未确定。针对ZO-2的一个独特区域制备了一种多克隆抗血清，并发现该抗血清能特异地标记MDCK质膜制剂中紧密连接的细胞质表面，表明ZO-2是紧密连接相关蛋白。全组织冰冻切片免疫组织化学染色显示，ZO-2定位于肝、肠、肾、睾丸和动脉内皮细胞的紧密连接区域，提示该蛋白是紧密连接的普遍成分。对非上皮性组织心脏冰冻切片进行的双标记免疫荧光显微镜显示，在粘连筋膜上存在ZO-1，但没有ZO-2染色，粘连筋膜是心肌细胞的一个特殊连接，以前已被证明含有ZO-1(Itoh，M.，S.Yonemura，A.Nagafuchi，S.Tsukita和Sh)。筑田。1991年。J.细胞生物学。115：1449-1462)。因此，ZO-2似乎不是筋膜粘连的一个组成部分，而且与ZO-1不同，该蛋白仅限于上皮紧密连接。
ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160- kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase- like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs- large-1 (dlg) gene of Drosophila, and a synapse-associated protein from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double- label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction.