Fresnedo O, De Heredia M L, Martínez M J, Cristóbal S, Rejas M T, Cuezva J M, Ochoa B
Department of Physiology, University of the Basque Country Medical School, Bilbao, Spain.
Hepatology. 2001 Mar;33(3):662-7. doi: 10.1053/jhep.2001.22763.
We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.
我们最近从大鼠肝脏微粒体中纯化出一种胆固醇酯水解酶（CEH）。针对纯化蛋白产生的抗体与一种106-kd的蛋白发生特异性反应，并中和了大鼠肝脏微粒体90%的CEH活性（《脂质研究杂志》1999年；40:715 - 725）。在这项研究中，我们使用抗CEH抗体来研究该蛋白在肝细胞中的亚细胞分布以及在大鼠中的组织特异性表达。对从分离的大鼠肝细胞获得的亚细胞组分进行蛋白质印迹分析显示，具有免疫反应性的106-kd CEH仅定位于微粒体中。该抗体还识别来自小鼠和人肝脏微粒体中的一种106-kd蛋白，但不识别来自大鼠非实质肝细胞微粒体中的该蛋白。对HepG2细胞进行共聚焦显微镜检查发现，CEH免疫反应性物质与内质网标志物钙连接蛋白共定位。此外，对大鼠肝脏薄片进行高分辨率免疫电子显微镜检查发现，CEH免疫反应性仅定位于肝细胞的内质网。在来自肾上腺、卵巢、睾丸、胰腺、肠道、白色脂肪组织、乳腺、肺、脾脏、大脑、主动脉和巨噬细胞的微粒体中未观察到CEH免疫反应性。我们报道了一种定位于哺乳动物肝细胞内质网的CEH，即内质网CEH（erCEH）。erCEH的亚细胞定位和组织限制性表达模式表明它可能在肝脏胆固醇代谢中具有独特功能。
