Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
Molecules. 2020 Apr 22;25(8):1942. doi: 10.3390/molecules25081942.
Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate β-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate β-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.
类胡萝卜素在自然界中含量丰富，是一类具有多烯四萜烯结构的脂溶性色素。它们具有抗氧化特性，人类摄入后会带来一定的健康益处。类胡萝卜素裂解加氧酶（CCOs）是一个氧化裂解类胡萝卜素的酶超家族，存在于所有生命领域。CCO 的进化非常复杂。例如，在这项研究中，我们偶然发现了一类新的真核 CCO，即脱辅基类胡萝卜素氧化酶样（ACOL）家族。该家族在动物基因组中有多个成员，并且缺乏动物特异性的氨基酸模体 PDPCK。这个模体可能与一些动物 CCO 的棕榈酰化有关。我们最近证明，类胡萝卜素加氧酶家族的两个哺乳动物成员视网膜色素上皮特异性 65kDa 蛋白（RPE65）和β-胡萝卜素加氧酶 2（BCO2）是棕榈酰化蛋白。在这里，我们使用酰基-resin-assisted capture (acyl-RAC) 方法来证明蛋白质的棕榈酰化，并使用免疫化学方法来定位 COS7 细胞系中缺乏和存在其底物 β-胡萝卜素时的小鼠 BCO2（mBCO2）。我们证明 mBCO2 的棕榈酰化依赖于进化保守的 PDPCK 模体，并且缺乏该模体的后生动物家族成员（文昌鱼β-胡萝卜素加氧酶样蛋白（BCOL）和 Acropora ACOL）不发生棕榈酰化。此外，我们观察到 mBCO2 的棕榈酰化状态及其膜结合依赖于其底物 β-胡萝卜素的存在。基于我们的结果，我们得出结论，大多数后生动物类胡萝卜素加氧酶保留了进化保守的棕榈酰化 PDPCK 模体，根据底物状态将蛋白质靶向到内部膜。例外情况是分泌的 BCOL 亚家族和严格的细胞质古 ACOL 类胡萝卜素加氧酶亚家族。
