Two carotenoid oxygenases contribute to mammalian provitamin A metabolism

Abstract

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15'-oxygenase (BCO1) and the β-carotene-9',10'-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15'-apocarotenoids, C20), whereas eccentric cleavage by BCO2 produces long-chain (>C20) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1(-/-), Bco2(-/-), and Bco1(-/-)Bco2(-/-) double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10'-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids.

Keywords: Carotenoid; Enzymes; Liver; Metabolism; Vitamin A.

FIGURE 1.

BC selectively accumulates in mice lacking BCO1. Six-week-old wild type (wt), Bco2^−/−, Bco1^−/−, and ko/ko mice were provided a controlled diet for 10 weeks with BC (50 mg/kg) serving as the sole source for β-apocarotenoid production. A, body weights at sacrifice. B, food intakes during the feeding period. C, weight curves of WT (diamonds), Bco2^−/− (squares), Bco1^−/− (triangles), and ko/ko (circles) mice. D, BC serum levels. E, hepatic BC levels. F, pulmonary BC levels. Values indicate means ± S.E. of at least five female animals per tissue and genotype. Statistical significance was determined by the two-tailed Student's t test with results compared with the WT group. Threshold of significance was set at *, p < 0.05.

FIGURE 2.

APO10ol is the major long-chain β-apocarotenoid in mouse liver. Six-week-old wild type (wt), Bco2^−/−, Bco1^−/−, and ko/ko mice were provided a controlled diet for 10 weeks with BC (50 mg/kg) as the sole source for β-apocarotenoid production. A, hepatic levels of free APO10ol (white bars) and total APO10ol (free + esterified) (dark gray bars). nd, not detectable. Values indicate means ± S.E. for at least five female animals per tissue and genotype. Statistical significance was tested by the two-tailed Student's t test with results compared with the WT group. Threshold of significance was set at p < 0.05. B and C, MS/MS diffraction patterns of APO10ol (B) and β-apo-10′-carotenal (APO10al) (C). D and E, presence of APO10ol (D) and APO10al (E) extracted from livers of Bco1^−/− (red trace) and ko/ko (blue trace) mice is indicated by an asterisk and identified by retention times and selected reaction monitoring (SRM) modes as compared with authentic standards (upper panel).

FIGURE 3.

β-Carotene derivative APO10ol can be esterified by LRAT. A, HPLC traces of AP010ol before and after incubation with RPE microsomes (15 μg of protein per assay). Peak 1 represents APO10ol esters and peak 2 nonesterified APO10ol. The insets reveal the spectral characteristics of peaks 1 and 2. B, time course of APO10ol esterification by RPE microsomes. The red curve represents the decrease in free APO10ol, and the black curve represents the increase of APO10ol esters. Values are expressed as concentrations of APO10ol and/or APO10ol esters divided by the total APO10ol concentration. C, RPE microsomes (15 μg of protein each) were preincubated with TLCK (1 mm) and or vehicle for only 10 min. Then APO10ol was added to a final concentration of 15 μm. After 15 min, reactions were stopped, and APO10ol esterification was measured. D, RPE microsomes (15 μg of protein per assay) were incubated in the presence of increasing amounts of APO10ol for 10 min. APO10ol ester formation was measured by HPLC analysis, and initial reaction velocities were plotted against the substrate concentration. V_max and K_m values for APO10ol were calculated with Origin 9 software (OriginLab). E, RPE microsomes (15 μg of protein per assay) were incubated with increasing amounts of ROL for 45 s. RE formation was measured by HPLC analysis, and initial reaction velocities were plotted against the substrate concentration. V_max and K_m values for ROL were calculated with Origin 9 software. In all tests for enzymatic activity, values represent means ± S.E. of three independent assays. F, 12-week-old WT and Lrat^−/− mice were gavaged with β-apo-10′-carotenal (20 mg/kg). Three hours later mice were sacrificed, and livers were isolated; lipids were extracted, and APO10ol esters were determined by HPLC analysis. Values present means ± S.E. of three animals per genotype. Statistical significance was tested by the two-tailed Student's t test with results compared with the WT group. Threshold of significance was set at p < 0.05.

FIGURE 4.

APO10ol transport and uptake. HepG2 cells were maintained at 37 °C in the presence (VAS) or absence (VAD) of vitamin A in the cell culture medium for 48 h. Cells then were incubated in 4 μm ROL or APO10ol with ethanol used as a vehicle control for 2 h. A, immunostaining for RBP (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. B, immunoblot analysis for RBP in protein extracts of HepG2 cells, using β-actin as the loading control. Ten μg of total protein were loaded per lane. C, 12-week-old Bco1^−/− mice were maintained on a VAD or VAS diet for 5 weeks. Then vitamin A-deprived mice were each gavaged with 30 mg/kg APO10ol or ROL dissolved in canola oil as a vehicle. Four hours later, mice were sacrificed; their livers were removed, and RBP levels were determined by immunoblot analysis. Ten μg of total protein were loaded per lane with β-actin used as a loading control. At least three mice were used per experimental group. D and E, spectral characteristics of purified ROL-hRBP (D) and APO10ol-hRBP (E). F, NIH3T3 cells expressing human LRAT and STRA6 were incubated in either 2 μm of ROL-hRBP (open circles), APO10ol-hRBP (open triangles), free ROL (filled circles), or free APO10ol (filled triangles). Uptake efficiency was measured as RE and APO10ol ester formation per μg of protein at different time points. Values shown for cell culture experiments indicate means ± S.E. from three independent experiments. Statistical significance was tested by the two-tailed Student's t test with results compared with free ROL (#) and APO10ol (*), respectively. Threshold of significance was set at p < 0.05.

FIGURE 5.

Hepatic vitamin A status of WT, Bco2^−/−, Bco1^−/−, and ko/ko mice. Six-week-old WT, Bco2^−/−, Bco1^−/−, and ko/ko mice were provided a 10-week controlled diet with BC (50 mg/kg) as the sole source for β-apocarotenoid production. A, hepatic RE levels. B, qRT-PCR analysis of hepatic Lrat mRNA levels. C, qRT-PCR analysis of hepatic Cyp26a1 mRNA levels. D, hepatic ROL levels. E, immunoblots for hepatic LRAT and RBP protein levels. β-Actin was used as loading control. F, serum ROL levels (upper panel) and serum RBP protein levels (lower panel). Ponceau S staining for albumin was used as loading control. Values indicate mean ± S.E. from 5 to 6 animals per genotype. Statistical significance compared with the WT group was tested by the two-tailed Student's t test. Threshold of significance was set at *, p < 0.05.

FIGURE 6.

Pulmonary vitamin A status of WT, Bco2^−/−, Bco1^−/−, and ko/ko mice. Six-week-old WT, Bco2^−/−, Bco1^−/−, and ko/ko mice were provided a 10-week controlled diet containing BC (50 mg/kg) as the sole source for β-apocarotenoid production. A, pulmonary RE levels. B, qRT-PCR analysis of pulmonary Lrat mRNA levels. C, qRT-PCR analysis of pulmonary Stra6 mRNA levels. D, pulmonary ROL levels. Values indicate means ± S.E. of results from 5 to 6 animals per genotype. Statistical significance compared with the WT group was tested by the two-tailed Student's t test. Threshold of significance was set at *, p < 0.05.

FIGURE 7.

Bco1 expression and hepatic vitamin A status of Bco2^−/− mice under different dietary conditions. A, hepatic Bco1 mRNA expression in female WT and Bco2^−/− mice provided a 10-week controlled diet with BC (50 mg/kg) as the sole source for β-apocarotenoid production. B and C, female WT and Bco2^−/− mice were provided a 10-week controlled diet with preformed vitamin A (4000 IU/kg). B, hepatic RE levels. C, qRT-PCR analyses of hepatic Bco1, Cyp26a1, and Lrat mRNA expression. Values indicate means ± S.E. from 5 to 6 animals per genotype. Statistical significance compared with the WT group was tested by the two-tailed Student's t test. Threshold of significance was set at *, p < 0.05.

FIGURE 8.

BCO1 converts long-chain β-apocarotenoids into retinoids. Recombinant human BCO1 was incubated with different β-apocarotenoids and BC (20 μm each) for 15 min. Lipophilic compounds were extracted and subjected to HPLC analyses. The panels show HPLC traces of substrates (blue traces) as compared with substrates incubated with recombinant human BCO1 (red traces). The insets give the spectral characteristics of different peaks. A, APO10ol; peak 1, all-trans-retinal-oxime (syn); peak 1′, all-trans-retinal-oxime (anti); peak 2, APO10ol. B, β-Apo-8′-carotenal; peak 3, β-apo-8′-carotenal; peak 4, all-trans-retinal. Note that apocarotenoids were not converted to the corresponding oxime because apo-8′-carotenal-oxime and all-trans-retinal-oxime (syn) co-eluted on the HPLC system. C, β-apo-12′-carotenoic acid; peak 1, all-trans-retinal-oxime peak (syn); peak 1′, all-trans-retinal-oxime (anti); peak 5, β-apo-12′-carotenoic acid. D, BC, peak 1, all-trans-retinal-oxime peak (syn); peak 1′, all-trans-retinal-oxime (anti); peak 6, BC.

FIGURE 9.

APO10ol is metabolized into retinoids by HepG2 cells. HepG2 cells were incubated with APO10ol (2 μm) (black line) or vehicle (ethanol) (gray line) for 12 h at 37 °C. Then cells were harvested, and lipophilic compounds were extracted and subjected to LC-MS analysis. A, analysis of nonpolar apocarotenoids. The presence of APO10ol, β-apo-10′-carotenal-oximes (APO10alox), ROL, and all-trans-retinal-oximes (RALox) extracted from HepG2 cells treated with APO10ol are indicated by asterisks and were identified by retention times and selected reaction monitoring modes as compared with authentic standards (upper panel). Esters of APO10ol and ROL are evident in examined samples by the presence of distinct peaks at 2.2 min of elution. B, analysis of acidic apocarotenoids. The presence of RA, β-apo-12′-carotenoic acid (APO12 acid), APO14 acid, β-apo-14′-carotenoic acid; APO10 acid, β-apo-10′-carotenoic acid extracted from HepG2 cells treated with APO10ol or vehicle are indicated by asterisks and were identified by retention times and selected reaction monitoring modes as compared with authentic standards (upper panel).

FIGURE 10.

BCO2 converts β-cryptoxanthin to β-apo-10′-apocarotenal. A, protein extract containing recombinant murine BCO2 was incubated with increasing concentrations of β-cryptoxanthin. Lipophilic compounds were extracted and subjected to HPLC analysis. Amounts of products (β-apo-10′-carotenal (filled diamonds) and 3-OH-β-apo-10′-carotenal (open squares)) are plotted against the substrate concentration. B, β-cryptoxanthin levels in the liver of 12-week-old female WT, Bco2^−/−, and Bco1^−/− mice. Values indicate means ± S.E. from three animals per genotype. Statistical significance compared with the WT group was tested by the two-tailed Student's t test with p < 0.05 considered significant.

FIGURE 11.

Proposed scheme for the metabolism of provitamin A carotenoids. β-Carotene is converted by BCO1 in the cytoplasm. β-Cryptoxanthin and other asymmetric carotenoids are transported to mitochondria. In mitochondria, BCO2 converts β-cryptoxanthin by oxidative cleavage at the C9,C10 double bond yielding β-apo-10′-carotenal and 3-hydroxy-β-ionone. β-Apo-10′-carotenal is reduced to the corresponding alcohol and transported to the cytoplasm by yet to be identified proteins as indicated by the question marks. β-Apo-10′-carotenol can then be esterified by LRAT and/or converted by BCO1 by oxidative cleavage to all-trans-retinal.