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The biochemical characterization of ferret carotene-9',10'-monooxygenase catalyzing cleavage of carotenoids in vitro and in vivo - PubMed

  • ️Sun Jan 01 2006

The biochemical characterization of ferret carotene-9',10'-monooxygenase catalyzing cleavage of carotenoids in vitro and in vivo

Kang-Quan Hu et al. J Biol Chem. 2006.

Abstract

Previous studies have shown that beta-carotene 15,15'-monooxygenase catalyzes the cleavage of beta-carotene at the central carbon 15,15'-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9',10'-monooxygenase (CMO2) in beta-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10'-carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9',10'-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of beta-carotene/lycopene cleavage into beta-apo-10'-carotenal/apo-10'-lycopenal in vitro and the formation of apo-10'-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-beta-carotene and the 5-cis- and 13-cis-isomers of lycopene at the 9',10'-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with beta-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10'-lycopenol, as well as up-regulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10'-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10'-lycopenoic acid and apo-10'-lycopenol, respectively, depending upon the presence of NAD+ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10'-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo-lycopenoids may have specific biological activities related to human health.

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Figures

FIGURE 1
FIGURE 1

Schematic illustration of a possible merabolic pathway of lycopene.

FIGURE 2
FIGURE 2. Comparison of the deduced amino acid sequences of the CMO2 among ferret, human, and mouse

Identity is indicated in yellow among three species and in blue between two species. The four conserved histidines (indicated by a single asterisk) and one glutamate (indicated in double asterisks) existed in the ferret CMO2, which has been demonstrated as putative iron-binding residues in all oxygenase family members (10). There CMO family signature sequence is underlined. The nucleotide sequence for the carotene 9′,10′-monooxygenase gene of ferret has been deposited in the GenBankTM with accession number AY527150.

FIGURE 3
FIGURE 3. Expression (left panel) and cytoplasmic localization (right panel) of ferret CMO2 in COS-1 cells

COS-1 cells were transfected with either an empty vector or expression vector encoding HA-tagged ferret CMO2 as described under “Experimental Procedures.” The lysates of transfected cells were analyzed by Western blotting with an antibody directed against the HA epitope tag encoded at the N terminus of each protein. For the localization of ferret CMO2 fusion protein, COS-1 cells on coverslips transfected with the expression vector encoding HA-tagged ferret CMO2 were fixed with 3.7% formaldehyde in PBS for 20 min and incubated with an anti-HA tag monoclonal antibody (Covance), followed by a green fluorescent secondary antibody. The images were viewed by fluorescence microscopy.

FIGURE 4
FIGURE 4. Tissue distribution of ferret CMO2

The relative levels (means ± S.D., n = 3) of CMO2 mRNA in various tissues of ferret were determined by real time PCR using brain as a reference organ and β-actin levels as a reference gene (upper panel ). The procedure for the real time PCR analysis was described under “Experimental Procedures.” Lower panel, the protein expression of CMO2 in ferret liver and testis was demonstrated by Western blot analysis using a purified polyclonal antibody, which is specifically recognized ferret CMO2 as described under “Experimental Procedures.” The HA-fCMO2 fusion protein from COS-1 cells was used as a positive control.

FIGURE 5
FIGURE 5. Expression of ferret CMO2 in SF9 insect cells and identification of cleavage product from β-carotene by HPLC analysis

Ferret CMO2 was expressed in SF9 insect cells by baculovirus as described under “Experimental Procedures.” Upper panel, the cell lysates from uninfected (lane 1) and ferret CMO2 baculovirus-infected (lane 2) insect cells were boiled in reducing sample buffer and subjected to 10% SDS-PAGE. The protein expression in insect cells can be detected by Coomassie Blue stain (A, upper panel), and the proteins were also transferred to polyvinylidene difluoride membrane and detected with CMO2-specific polyclonal antibody (B, upper panel). Lower panel, HPLC profile of the cleavage products of all-trans-β-carotene by ferret CMO2 enzyme. All-trans-β-carotene (3 μM) was incubated with the homogenates from either uninfected (A) or ferret CMO2 baculovirus-infected (B) insect cells for 1 h at 37 °C as described under “Experimental Procedures.” The cleavage products extracted from the incubation mixture were separated by reverse phase HPLC using a C18 column. A peak corresponding to authentic β-apo-10′-carotenal standard (C) was detected at 450 nm only in the incubation mixture with the homogenates of ferret CMO2 baculovirus-infected cells (B) but not in that of uninfected cells (A). D, spectral analysis of the cleavage product (blue line) of all-trans-β-carotene versus β -apo-10′-carotenal standard (red line). Both the retention time and absorption spectrum of the product from all-trans-β-carotene matched exactly with that of standard β-apo-10′-carotenal.

FIGURE 6
FIGURE 6. Effects of pH, time, protein, and substrate concentrations on β-apo-10′-carotenal productions by ferret CMO2

A, pH optimum of the enzyme was detected at the indicated pH value in the presence of 6 mg of homogenate protein containing ferret CMO2 and 3 μM all-trans-β-carotene at 37 °C for 1 h; B, all-trans-β-carotene (3 μ M) was incubated with 6 mg of homogenate expressing ferret CMO2 at 37 °C for various time points; C, the incubations were carried out using various protein concentrations of cell homogenates expressing ferret CMO2 with 3 μM all-trans-β-carotene at 37° for 1 h; and D, the effect of substrate concentration was conducted with homogenates of insect cells expressing ferret CMO2 with various all-trans-β-carotene concentration. The enzyme reactions were conducted in a volume of 1 ml of assay buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM KCl, 10 μM Fe2SO2, 3 mM NAD, 0.3 mM DTT, and 6 mg of protein of the insect cell homogenates expressing ferret CMO2 at 37 °C for 60 min, otherwise as indicated on the figures. The procedures for the incubation, extraction, and HPLC analysis were described under “Experimental Procedures.” The data are the average of two independent experiments. The variation in the data between the two experiments was less than 3% of the data average.

FIGURE 7
FIGURE 7. HPLC profile of the cleavage products of lycopene by ferret CMO2 enzyme

The assays were carried out using the homogenates from CMO2 baculovirus-infected cells and incubated with either all-trans-lycopene (97%) (A), or 5-cis-lycopene (38%) (B), or 13-cis-lycopene (29%) (C ) at 37 °C for 60 min. The cleavage products extracted from the incubation mixture were separated by reverse phase HPLC using a C30 column as described under “Experimental Procedures.” D, the HPLC pattern of an authentic apo-10′-lycopenal standard matches exactly both the retention time (B, peak 1 and E) and the absorption spectrum of the product from 5-cis-lycopene. E, spectral analysis of the cleavage products of lycopene isomers. The absorption spectrum of the apo-10′-lycopenal standard (black line, peak 1 from D) compared with peak 1 (blue line, the reaction product in B) and peak 2 (red line, the reaction product in C ) extracted from the incubation of the homogenates of insect cells expressing ferret CMO2 with lycopene isomers. F, spectra of lycopene isomers (black line, all-trans-lycopene; blue line, 5-cis-lycopene; and red line, 13-cis-lycopene). The procedures for the incubation, extraction, and HPLC analysis were as described under “Experimental Procedures.”

FIGURE 8
FIGURE 8. Effects of pH, time, protein, and substrate concentrations on the production of apo-10′-lycopenal from lycopene (20% as cis-isomers) with ferret CMO2

A, the reaction as a function of pH was conducted with homogenates of insect cells expressing ferret CMO2 with 5 μMlycopene. B, the time course of the reaction was conducted with homogenates of insect cells expressing ferret CMO2 with 5 μM lycopene for various time points. C, the reaction as a function of protein concentration was conducted with various protein concentrations of insect cells expressing ferret CMO2 with 5 μM lycopene. D, the reaction as a function of substrate concentration was conducted with homogenates of insect cells expressing ferret CMO2 with various lycopene concentrations. The enzyme reaction was conducted in a volume of 1 ml of assay buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM KCl, 10 μM Fe2SO2, 3 mM NAD, 0.3 mM DTT, and the 6 mg of protein of insect cell homogenates expressing ferret CMO2 at 37 °C for 60 min, unless indicated. The procedures for the incubation, extraction, and HPLC analysis were as described under “Experimental Procedures.” The data are the average of two independent experiments. The variation in the data between the two experiments was less than 9% of the data average.

FIGURE 9
FIGURE 9. HPLC of lycopene metabolites in the lungs of ferrets after 9 weeks of supplementation with lycopene

A, HPLC pattern of standard apo-10′-lycopenol. B, HPLC pattern of ferret lung tissue extract after supplementation with placebo. C, HPLC pattern of ferret lung tissue extract after supplementation with lyco-pene. D, spectral analysis of the metabolic product (1st peak) of lycopene in the lung of ferret. The absorption spectrum of an apo-10′-lycopenol standard (boldface line) matched exactly that of the unknown compound (C, peak 1, pale line) extracted from ferret lung tissue after supplementation with lycopene. The peak identifications are as follows: 1, apo-10′-lycopenol; 2, echinenone (internal standard); 3, 13-cis-lycopene; 4, 9-cis-lycopene; 5, all-trans-lycopene; and 6, 5-cis-lycopene. The procedures for the animal experiment, tissue extraction, and HPLC analysis were as described under “Experimental Procedures.”

FIGURE 10
FIGURE 10. Expression of CMO2 in the lungs of ferrets after lycopene supplementation for 9 weeks

The relative levels (means ± S.D.) of CMO2 mRNA in lungs of ferrets were determined by real time PCR using β-actin levels as a reference gene. The procedures for the animal experiment, lung mRNA extraction, and real time PCR method are described under “Experimental Procedures.” Six ferrets were in each treatment group (Control, no treatment; Lycopene, lycopene-supplemented). * indicates significantly different at p < 0.05.

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