Palmitoylation of Metazoan Carotenoid Oxygenases

Abstract

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.

Xanthophylls Modulate Palmitoylation of Mammalian β-Carotene Oxygenase 2

“…We did not observe any colocalization of mBCO2 with other organelles ( Figure 3 A, bottom left panel, and 4B) as indicated by their low correlation coefficient score compared with mitochondrial localization. Our data are in full agreement with previous results for human BCO2 [ 21 , 34 ]. However, upon addition of substrates to mBCO2 we observed that a fraction of mBCO2 colocalizes with the nuclear DAPI stain and the colocalization with mitochondrial markers is significantly decreased ( Figure 3 A, top right panel, C).…”

“…We previously established that mBCO2 in eukaryotic HEK293F cells is palmitoylated. We discovered that in the presence of the mBCO2 substrate β-carotene mBCO2 loses palmitoylation [ 34 ]. Knowing that BCO2 cleaves xanthophylls [ 17 , 48 , 49 ] we decided to run palmitoylation assays to study the palmitoylation status of mBCO2 in the presence of xanthophylls.…”

“…Mouse BCO2 cDNAs were prepared as described [ 34 ] and used to generate vectors for the expression of BCO2 in mammalian cells. Briefly, BCO2 was subcloned into the bicistronic expression vector pVitro2 (InvivoGen, San Diego, CA, USA) and into the Gateway cloning vector pcDNA6.2c-Lumio-DEST vector (Thermo Fisher (Invitrogen), Carlsbad, CA, USA) to generate untagged or C-terminal V5/lumio tagged versions of mouse BCO2, respectively.…”

“…For cell lysis and the Acyl-RAC assay, we followed the same protocol as previously described in detail, with slight modifications [ 34 , 36 ]. BCO2-overexpressing HEK293F cells were washed with 1X-PBS and resuspended in lysis buffer (50 mM HEPES (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1% glycerol, and 1X complete protease inhibitor cocktail (Roche Diagnostics)).…”

“…BCO2-overexpressing COS7 cells (1 × 10 6 cells/mL) seeded on poly L-lysine coated 18 mm coverslips were analyzed by immunofluorescence microscopy to determine the localization of BCO2 protein in different subcellular organelles as described previously [ 34 ]. Briefly, COS7 cells were transfected with 20 µg of BCO2-Lumio V5 tag plasmid using Fugene ® 6 transfection reagent (1:6 DNA: Fugene 6 ratio).…”