Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells
Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells
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DOI：
10.1074/jbc.m310042200
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发表时间：
2004-01-02
影响因子：
4.8
通讯作者：
Travis, GH
中科院分区：
文献类型：
--
作者：
Mata, NL;Moghrabi, WN;Travis, GH
视紫红质色素分子的光子俘获诱导其视黄醛发色团的11-顺式全反式异构化。为了恢复对光的敏感性，全反式视黄醛必须通过一种称为视觉周期的酶途径进行化学再异构化。RPE65是视网膜色素上皮(RPE)细胞中的一种丰富的蛋白质，也是β-胡萝卜素双加氧酶的同系物，似乎在这一途径中发挥了作用。RPE65(-/-)基因敲除小鼠大量积累全反式视黄酸酯，但视网膜中缺乏11顺式维甲酸和视紫红质视觉色素。人类RPE65基因的突变会导致一种名为莱伯氏先天性黑发的严重隐性失明疾病。然而，RPE65的功能尚不清楚。在这里，我们证明了RPE65通过光谱移位实验、凝胶过滤中的共洗脱和免疫共沉淀，特异性地结合了全反式视黄酸棕榈酸酯，但不能结合11-顺式视黄酸棕榈酸酯。利用一种新的脂质体荧光共振能量转移(FRET)结合实验，我们证明了RPE65从磷脂膜中提取全反式视黄酸酯。异构酶活性测定表明，RPE65能强烈促进牛RPE细胞微粒体中全反式视黄酸棕榈酸酯转化为11-顺式视黄醇。此外，我们还发现，在RPE65(-/-)小鼠的膜上加入RPE65后，异构酶活性恢复到野生型水平，而RPE65(-/-)小鼠的膜上没有检测到异构酶活性。然而，RPE65本身并不具有固有的异构酶活性。这些观察结果表明，RPE65提供视黄酸酯作为异构酶的底物，用于合成视觉生色团。这一被提出的功能解释了缺乏RPE65的小鼠和人类的表型。
Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65(-/-) knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer ( FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65(-/-) mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.