Mata Nathan L, Moghrabi Walid N, Lee Jung S, Bui Tam V, Radu Roxana A, Horwitz Joseph, Travis Gabriel H
Jules Stein Eye Institute, University of California School of Medicine, Los Angeles, California 90095, USA.
J Biol Chem. 2004 Jan 2;279(1):635-43. doi: 10.1074/jbc.M310042200. Epub 2003 Oct 7.
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.
视紫红质色素分子捕获光子会诱导其视黄醛发色团从11-顺式异构化为全反式。为恢复光敏感性，全反式视黄醛必须通过一种称为视觉循环的酶途径进行化学重新异构化。Rpe65是视网膜色素上皮（RPE）细胞中一种丰富的蛋白质，也是β-胡萝卜素双加氧酶的同源物，似乎在该途径中发挥作用。Rpe65基因敲除小鼠大量积累全反式视黄酯，但视网膜中缺乏11-顺式视黄醛类和视紫红质视觉色素。人类RPE65基因突变会导致一种严重的隐性致盲疾病，称为莱伯先天性黑矇。然而，Rpe65的功能尚不清楚。在这里，我们通过光谱位移分析、凝胶过滤过程中的共洗脱以及共免疫沉淀表明，Rpe65特异性结合全反式视黄酯棕榈酸酯，而不结合11-顺式视黄酯棕榈酸酯。使用脂质体中的新型荧光共振能量转移（FRET）结合分析，我们证明Rpe65从磷脂膜中提取全反式视黄酯。异构酶活性分析表明，Rpe65强烈刺激牛RPE细胞微粒体中全反式视黄酯棕榈酸酯向11-顺式视黄醇的酶促转化。此外，我们表明，将Rpe65添加到来自Rpe65基因敲除小鼠的膜中（这些膜没有可检测到的异构酶活性），可将异构酶活性恢复到野生型水平。然而，Rpe65本身没有内在的异构酶活性。这些观察结果表明，Rpe65将视黄酯作为底物提供给异构酶，用于合成视觉发色团。这一提出的功能解释了缺乏Rpe65的小鼠和人类的表型。
