Kinouchi Rieko, Kinouchi Tadatoshi, Hamamoto Toshirou, Saito Takakazu, Tavares Adriano, Tsuru Tadahiko, Yamagami Satoru
Department of Ophthalmology, Jichi Medical School, Tochigi, Japan.
Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1397-403. doi: 10.1167/iovs.05-0602.
The gene expression profile of human corneal endothelium (CE) was established with the gene signature system. A novel gene, GS3582, was abundantly transcribed in the CE compared with other tissues according to a human gene expression database. This protein was designated corneal endothelium-specific protein (CESP)-1. The tissue distribution and subcellular localization of CESP-1 was assessed in humans and mice, to investigate its physiological function.
Rabbit and mouse CESP-1 cDNAs were cloned, and a polyclonal anti-human CESP-1 antibody (Ab) and anti-mouse N- or C-terminal ovary-specific acidic protein (OSAP)-1 Ab were produced. CESP-1 expression was investigated in human and mouse corneas by Western blot and/or immunohistochemical analysis. The distribution of CESP-1 in human tissues was also examined by Western blot analysis. To identify the subcellular localization of CESP-1, cultured human CE was colabeled with anti-human CESP-1 Ab and anti-cytochrome c monoclonal Ab or anti-GRP78 monoclonal Ab for confocal microscopy.
The rabbit and mouse CESP-1 cDNA sequences contained an open reading frame coding 242 and 283 amino acids, respectively. Mouse CESP-1 was entirely consistent with mouse OSAP. Western blot analysis showed that CESP-1 was expressed in the human corneal epithelium, CE, cultured CE, brain, testis, and ovary. Mouse CESP-1 was also expressed in mouse corneal epithelium and CE with anti-mouse C- but not N-terminal OSAP Ab according to immunohistochemical analysis. Subcellular localization of CESP-1 to the mitochondria was demonstrated in cultured human CE. The N-terminal of CESP-1, possessing a mitochondrial targeting sequence, may be processed after the protein is imported into the mitochondria.
CESP-1 was distributed in the corneal epithelium, the CE and cultured human CE, as well as the brain, testis, and ovary. CESP-1 was localized in the mitochondria of cultured human CE. These findings may provide some clues about the physiological function of CESP-1.
利用基因特征系统建立人角膜内皮（CE）的基因表达谱。根据人类基因表达数据库，与其他组织相比，一种新基因GS3582在CE中大量转录。该蛋白被命名为角膜内皮特异性蛋白（CESP）-1。评估CESP-1在人和小鼠中的组织分布及亚细胞定位，以研究其生理功能。
克隆兔和小鼠CESP-1的cDNA，并制备多克隆抗人CESP-1抗体（Ab）以及抗小鼠N端或C端卵巢特异性酸性蛋白（OSAP）-1抗体。通过蛋白质印迹法和/或免疫组织化学分析研究CESP-1在人和小鼠角膜中的表达。还通过蛋白质印迹分析检测CESP-1在人体组织中的分布。为确定CESP-1的亚细胞定位，对培养的人CE用抗人CESP-1抗体和抗细胞色素c单克隆抗体或抗GRP78单克隆抗体进行共标记，用于共聚焦显微镜观察。
兔和小鼠CESP-1的cDNA序列分别包含一个编码242和283个氨基酸的开放阅读框。小鼠CESP-1与小鼠OSAP完全一致。蛋白质印迹分析表明，CESP-1在人角膜上皮、CE、培养的CE、脑、睾丸和卵巢中表达。免疫组织化学分析显示，用抗小鼠C端而非N端OSAP抗体检测时，小鼠CESP-1也在小鼠角膜上皮和CE中表达。在培养的人CE中证实CESP-1定位于线粒体。CESP-1的N端具有线粒体靶向序列，可能在蛋白质导入线粒体后被加工。
CESP-1分布于角膜上皮、CE和培养的人CE以及脑、睾丸和卵巢中。CESP-1定位于培养的人CE的线粒体中。这些发现可能为CESP-1的生理功能提供一些线索。
