Cavaney-Brooker D M, Rakoczy P E
Centre for Ophthalmology and Visual Science, Lions Eye Institute, University of Western Australia, Nedlands, Australia.
Curr Eye Res. 1999 Apr;18(4):310-8. doi: 10.1076/ceyr.18.4.310.5357.
It has been proposed that the major proteolytic enzyme responsible for the proteolysis of photoreceptor outer segments is an aspartic protease similar or identical to cathepsin D. The aim of this study was to determine if the major retinal pigment epithelial (RPE) aspartic protease was cathepsin D, to study its distribution and investigate its transcription start sites (TSS).
An RPE cDNA library was screened at low stringency for the presence of aspartic proteases. DNA sequencing was used to analyze the identified clones. The expression of cathepsin D mRNA was analyzed by Northern blot while immunohistochemistry was used to demonstrate cathepsin D related immunoreactivity in an eye section. RNase protection assay was used to map the Cathepsin D TSS in RPE cells.
The aspartic protease identified in the RPE cDNA library was identical to the DNA sequence of cathepsin D found in other tissues. Northern blot analysis of a wide range of cell types showed that the highest level of cathepsin D mRNA expression was found in RPE cells which was similar only to cathepsin D expression in MCF7 breast cancer cells. Immunohistochemical staining of a human retina confirmed the inherent nature of high cathepsin D expression in RPE cells. An RNase protection assay demonstrated two major cathepsin D TSS in RPE cells. One of them was identical to the TSS responsible for the constitutive expression of cathepsin D and the other, a TATA box-controlled TSS, was identical to a TSS found in MCF7 cells. Cathepsin D expression was not enhanced by the phagocytosis and digestion of rod outer segments (ROS) and ROS phagocytosis did not induce the use of other cathepsin D TSS in the RPE cells.
The major aspartic protease identified in RPE cells was cathepsin D. In addition, it was demonstrated that the high level of cathepsin D expression in RPE cells is the intrinsic nature of these cells and that it is linked to a TATA box-controlled TSS. This TSS has previously been described as an estrogen regulated TSS and further studies are required to identify the RPE-specific inducer or indirect factors that may be responsible for the enhanced expression of cathepsin D in the RPE cells.
有人提出，负责光感受器外段蛋白水解的主要蛋白水解酶是一种与组织蛋白酶D相似或相同的天冬氨酸蛋白酶。本研究的目的是确定视网膜色素上皮（RPE）主要的天冬氨酸蛋白酶是否为组织蛋白酶D，研究其分布并探究其转录起始位点（TSS）。
以低严谨度筛选RPE cDNA文库中是否存在天冬氨酸蛋白酶。使用DNA测序分析鉴定出的克隆。通过Northern印迹分析组织蛋白酶D mRNA的表达，同时使用免疫组织化学在眼切片中显示组织蛋白酶D相关的免疫反应性。使用核糖核酸酶保护试验来定位RPE细胞中组织蛋白酶D的TSS。
在RPE cDNA文库中鉴定出的天冬氨酸蛋白酶与在其他组织中发现的组织蛋白酶D的DNA序列相同。对多种细胞类型的Northern印迹分析表明，组织蛋白酶D mRNA表达水平最高的是RPE细胞，这仅与MCF7乳腺癌细胞中组织蛋白酶D的表达相似。人视网膜的免疫组织化学染色证实了RPE细胞中组织蛋白酶D高表达的内在特性。核糖核酸酶保护试验在RPE细胞中显示出两个主要的组织蛋白酶D TSS。其中一个与负责组织蛋白酶D组成型表达的TSS相同，另一个由TATA盒控制的TSS与在MCF7细胞中发现的一个TSS相同。杆状外段（ROS）的吞噬和消化不会增强组织蛋白酶D的表达，并且ROS吞噬不会诱导RPE细胞中使用其他组织蛋白酶D TSS。
在RPE细胞中鉴定出的主要天冬氨酸蛋白酶是组织蛋白酶D。此外，已证明RPE细胞中组织蛋白酶D的高表达是这些细胞的内在特性，并且它与由TATA盒控制的TSS相关。该TSS先前已被描述为雌激素调节的TSS，需要进一步研究以鉴定可能导致RPE细胞中组织蛋白酶D表达增强的RPE特异性诱导剂或间接因素。
