Unveiling BCO2 function in macular pigment metabolism: Mitochondrial processing and expression in the primate retina

Abstract

BCO2 (β-carotene oxygenase 2) converts carotenoids into apocarotenoids by oxidative cleavage across double bonds and controls carotenoid homeostasis in vertebrate tissues. In this study, we examined BCO2's expression, localization, and activity in human cell lines and the retina. We generated peptide antibodies directed against primate BCO2 and validated their specificity using recombinant BCO1 (β-carotene oxygenase 1) and BCO2 proteins expressed in bacteria. The antibodies specifically detected human BCO2 by Western blot. In BCO2 expressing HepG2 cells, the antibodies recognized a 65 kDa mitochondrial protein that co-migrated with a recombinant truncated 522-amino-acid BCO2 variant, suggesting post-translational processing of the 579 amino acid long human BCO2 protein. Immunohistochemical analysis of macaque retina sections revealed BCO2 localization in the retinal pigment epithelium, photoreceptor inner segments, plexiform layer, and ganglion cell layer. Co-staining with COX IV indicated a mitochondrial localization of retinal BCO2 within photoreceptor inner segments. Western blot analysis of human donor retinas, separated into central and peripheral regions, identified higher BCO2 expression in the peripheral retina. Enzymatic activity assays demonstrated that BCO2 interacted with Aster proteins that transport carotenoids within cells. Our studies establish BCO2 as a mitochondrial protein expressed in the primate retina, where it likely plays a pivotal role in the metabolism of macular pigments and the maintenance of retinal health.