Wang Cheng, JeBailey Lellean, Ridgway Neale D
Atlantic Research Center, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Biochem J. 2002 Feb 1;361(Pt 3):461-72. doi: 10.1042/0264-6021:3610461.
Oxysterol-binding protein (OSBP) is the prototypical member of a class of phospholipid and oxysterol-binding proteins that interacts with the Golgi apparatus and regulates lipid and cholesterol metabolism. As a result of recent sequencing efforts, eleven other OSBP-related proteins (ORPs) have been identified in humans. We have investigated the structure, oxysterol-binding activity, cellular localization and function of ORP4 (also designated OSBP2 or HLM), a homologue that shares the highest degree of similarity with OSBP. Two ORP4 cDNAs were identified: a full-length ORP4 containing a pleckstrin homology (PH) domain and an oxysterol-binding region (designated ORP4-L), and a splice variant in which the PH domain and part of the oxysterol-binding domain were deleted (designated ORP4-S). ORP4 mRNA and protein expression overlapped partially with OSBP and were restricted to brain, heart, muscle and kidney. Like OSBP, ORP4-L bound [3H]25-hydroxycholesterol with high affinity and specificity. In contrast, ORP4-S did not bind [3H]25-hydroxycholesterol or [3H]7-ketocholesterol. Immunofluorescence localization in stably transfected Chinese hamster ovary cells showed that ORP4-S co-localized with vimentin and caused the intermediate filament network to bundle or aggregate. ORP4-L displayed a diffuse staining pattern that did not overlap with vimentin except when the microtubule network was disrupted with nocodazole. Oxysterols had no effect on the localization of either ORP4. Cells overexpressing ORP4-S had a 40% reduction in the esterification of low-density-lipoprotein-derived cholesterol, demonstrating that ORP4 interaction with intermediate filaments inhibits an intracellular cholesterol-transport pathway mediated by vimentin. These studies elucidate a hitherto unknown relationship between OSBPs and the intermediate filament network that influences cholesterol transport.
氧化甾醇结合蛋白(OSBP)是一类磷脂和氧化甾醇结合蛋白的典型成员,它与高尔基体相互作用并调节脂质和胆固醇代谢。近期的测序研究结果显示,在人类中已鉴定出另外11种与OSBP相关的蛋白(ORPs)。我们研究了ORP4(也称为OSBP2或HLM)的结构、氧化甾醇结合活性、细胞定位和功能,ORP4是与OSBP相似度最高的同源物。鉴定出了两个ORP4 cDNA:一个全长ORP4,含有一个普列克底物蛋白同源(PH)结构域和一个氧化甾醇结合区域(称为ORP4-L),以及一个剪接变体,其中PH结构域和部分氧化甾醇结合结构域被删除(称为ORP4-S)。ORP4 mRNA和蛋白表达与OSBP部分重叠,且局限于脑、心脏、肌肉和肾脏。与OSBP一样,ORP4-L以高亲和力和特异性结合[3H]25-羟基胆固醇。相比之下,ORP4-S不结合[3H]25-羟基胆固醇或[3H]7-酮胆固醇。在稳定转染的中国仓鼠卵巢细胞中的免疫荧光定位显示,ORP4-S与波形蛋白共定位,并导致中间丝网络成束或聚集。ORP4-L呈现出弥散的染色模式,除了用诺考达唑破坏微管网络时,它与波形蛋白不重叠。氧化甾醇对两种ORP4的定位均无影响。过表达ORP4-S的细胞中,低密度脂蛋白衍生胆固醇的酯化减少了40%,这表明ORP4与中间丝的相互作用抑制了由波形蛋白介导的细胞内胆固醇转运途径。这些研究阐明了OSBPs与影响胆固醇转运的中间丝网络之间迄今未知的关系。