Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases

Article abstract of DOI:10.1016/j.steroids.2006.04.009

vitamin D is 25-hydroxylated in the liver, before being activated by 1α-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D₃ 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D₃ (turnover number, 0.087 min^-1), vitamin D₂ (0.16 min^-1), and 1α-hydroxyvitamin D₃ (2.2 min^-1). Interestingly, human CYP2J2 hydroxylated vitamin D₂, an exogenous vitamin D, at a higher rate than it did vitamin D₃, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D₃ (1.4 min^-1) more efficiently than vitamin D₂ (0.86 min^-1). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D₃ and D₂.

Full text of DOI:10.1016/j.steroids.2006.04.009

steroids 7 1 ( 2 0 0 6 ) 849–856
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Characterization of rat and human CYP2J enzymes as
Vitamin D 25-hydroxylases
Isamu Aibaa, Tomoaki Yamasakia, Toshimasa Shinkib, Shunsuke Izumia, Keiko
Yamamotoc, Sachiko Yamadac, Hiroaki Teratoa, Hiroshi Idea, Yoshihiko Ohyamaa,∗
a
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima 739-8526, Japan
b
Department of Biochemistry, Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Saitama 362-0806, Japan
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
c
2-3-10 Kanda-Surugadai, Chiyoda-Ku, Tokyo 101-0062, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
vitamin D is 25-hydroxylated in the liver, before being activated by 1␣-hydroxylation in the
kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vita-
min D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a
novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848–56]. In this
study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat
CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in
Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found signif-
icant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min−1), vitamin
Received 21 March 2005
Accepted 15 April 2005
Published on line 13 July 2006
Keywords:
vitamin D3
vitamin D2
25-Hydroxylase
CYP2J2
D
2 (0.16 min−1), and 1␣-hydroxyvitamin D3 (2.2 min−1). Interestingly, human CYP2J2 hydrox-
ylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an
endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min−1) more effi-
ciently than vitamin D2 (0.86 min−1). Our study demonstrated that human CYP2J2 exhibits
25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is
weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3
and D2.
CYP2J3
Abbreviations:
25(OH)D3, 25-hydroxyvitaimin D3;
1,25(OH)2D3, 1,25-dihydroxyvitamin
D3; 1␣(OH)D3, 1␣-hydroxyvitamin
D3; CYP, cytochrome P450; HPLC,
high performance liquid
© 2006 Elsevier Inc. All rights reserved.
chromatography; PCR, polymerase
chain reaction; PAGE,
polyacrylamide gel electrophoresis
1.
Introduction
conversion of 7-dehydrocholesterol in the skin or from the
diet, while vitamin D2 derived from a plant sterol, ergos-
Two vitamin D compounds, vitamin D3 and vitamin D2, are
known to be physiologically important for calcium homeosta-
sis [1–5]. vitamin D3 can be obtained by the light and heat
terol, can only be obtained from the diet. A recent report
showed vitamin D2 is much less effective than vitamin
D3 in humans [6]. Both vitamin D3 and D2 are converted
∗
Corresponding author. Tel.: +81 82 424 7458; fax: +81 82 424 7458.
E-mail address: ohyama@sci.hiroshima-u.ac.jp (Y. Ohyama).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.04.009

850
steroids 7 1 ( 2 0 0 6 ) 849–856
into the active forms, 1,25(OH)2D3 and 1,25(OH)2D2, respec-
tively, in the body. vitamin D2 has an additional active form,
1,24(OH)2D2, which has been reported to be physiologically
less active, but less toxic than 1,25(OH)2D2 and 1,25(OH)2D3
[7,8]. In both vitamin D compounds, the hydroxylation of the
side chain at C-25 or C-24 is a prerequisite for the subse-
quent 1␣-hydroxylation controlled by physiological conditions
[3,9,10]. Therefore, characterization of the enzymes involved
in the side chain hydroxylation of vitamin D is important
for understanding the whole scheme of vitamin D activa-
tion.
To date, six vitamin D 25-hydroxylases have been reported:
CYP27A1, CYP2R1, CYP3A4, CYP2C11, CYP2D25, and CYP2J3
[11,12]. CYP27A1 is a mitochondrial P450, hydroxylating at C-
25 of vitamin D3 and at C-24 of vitamin D2, as well as at
C-27 of the intermediates of bile acid synthesis [13,14]. The
CYP27A1 gene is highly conserved in all vertebrates. How-
ever, knockout mice of the CYP27A1 gene exhibit no signif-
icant changes in vitamin D metabolism. Also, cerebrotendi-
nous xanthomatosis patients, who have serious mutations in
the CYP27A1 gene, exhibit no or little changes in the serum
concentration of 25-hydroxyvitamin D compounds in spite of
abnormal cholesterol metabolism [15,16]. Therefore, CYP27A1
is thought to be critical for bile acid synthesis as a sterol 27-
hydroxylase.
2.
Experimental procedures
2.1.
Materials
vitamin D3 was obtained from Katayama Chemical Co.
(Osaka, Japan). vitamin D2 was obtained from Fluca (Ger-
many). 1␣,25(OH)2D3, 25(OH)D3, 1␣(OH)D3, isopropyl-1-thio-
␤-d-galactoside, and cholic acid were purchased from Wako
Pure Chemical Industries (Osaka, Japan). ␦-Aminolevulinic
acid was obtained from Sigma, and ␤-NADPH was obtained
from Oriental Co. (Tokyo, Japan). Emulgen 911 was supplied by
Kao Co. (Tokyo, Japan). A pGro7 expression vector that con-
tained the GroEL–GroES gene was obtained from Takara Bio
Inc. (Otsu, Japan). Restriction enzymes were obtained from
New England Biolabs Inc. The expression plasmid, pKSN2, was
modified from pkk223-3 as previously described by Akiyoshi-
Shibata et al. [25]. 25(OH)D2 was synthesized as described
[26]. Primers for polymerase chain reactions (PCR) were syn-
thesized by Kurabo Industries Ltd. (Osaka, Japan). All other
chemicals were of the highest quality commercially avail-
able.
2.2.
Cloning of CYP2J2 cDNA and construction of
N-terminal deletion mutants
Recently, CYP2R1 has been identified as a novel microso-
mal vitamin D 25-hydroxylase by expression cloning using a
CYP27A1-knockout mouse cDNA library [17]. The CYP2R1 gene
is conserved in all vertebrates, and is dominantly expressed
in the liver and testes of mice. The following study of Cheng
et al. revealed that a patient with low circulating levels of 25-
hydroxyvitamin D is homozygous for a mutation of CYP2R1
gene [18]. These findings have established the CYP2R1 is the
essential vitamin D 25-hydroxylase in humans.
CYP3A4 is the most abundant P450 expressed in the adult
human liver and intestine, and is involved in the metabolism
of about half of the drugs in use. Interestingly, it exhibits
25-hydroxylation activity for vitamin D2, but not for vita-
min D3 [19]. CYP2C11 and CYP2D25 have been purified as
major microsomal vitamin D 25-hydroxylases in male rats
and pigs, respectively [20–22]. CYP2C11 is a P450 specific
to the male rat. In spite of the significant 25-hydroxylation
activity of CYP2D25, its human ortholog, CYP2D6, shows no
detectable effect on vitamin D [23]. Thus, CYP2C11, CYP2D25,
and CYP3A4 seem to be species-specific vitamin D hydroxy-
lases.
We have recently identified CYP2J3 as a novel vitamin D3
25-hydroxylase in rats [24]. CYP2J3 is highly expressed in the
liver regardless of sex, and exhibits markedly higher (about
10 times) 25-hydroxylation activity toward vitamin D3 than
do other 25-hydroxylases, such as CYP2C11, CYP2D25, and
CYP27A1. Therefore, it is of interest to determine whether
human CYP2J2, the sole human CYP2J subfamily member with
73% amino acid homology to CYP2J3, shows 25-hydroxylation
activity even though CYP2R1 is the essential 25-hydroxylase
in humans. Here, we prepared a recombinant human CYP2J2,
and compared its catalytic activity toward vitamin D with that
of rat CYP2J3. We found that CYP2J2 shows significant 25-
hydroxylation activity, which suggests the contribution of the
CYP2J subfamily to vitamin D metabolism.
CYP2J2 cDNA (GenBank accession no. U37143) was cloned
from a Cap Site cDNA library of the human liver (Wako
Pure Chemical Industries) by PCR using the following primers
containing an extra NdeI or HindIII site for ligation into the
expression vector: forward primer (IA-1), 5ꢀ-CAT ATG GCT
GCA GCA ATG GGC TCT CTG GCG-3ꢀ and reverse primer
(IA-2), 5ꢀ-AAG CTT TCA GAA CAC GTG CCA TGT C-3ꢀ. The
second codon was changed from wild type Leu (CTC) to
Ala (GCT) in the primer IA-1. Two deletion constructs of N-
terminal regions (D19 lacking 2-19 amino acid residues, and
D34 devoid of 2-34) of CYP2J2 were prepared by PCR using
the following forward primers: 5ꢀ-CAT ATG GCT CTG TTA
TTA GCA ACT GTC GCC-3ꢀ for D19 mutant, and 5ꢀ-CAT ATG
GCT AAA CGT CGT CGC CCA AAG AAC TAC CCG-3ꢀ for D34
mutant, together with the IA-2 reverse primer using an iso-
lated CYP2J2 cDNA as the template. Typical PCR parameters
were: 95 ◦C for 2 min, 35 cycles of 95 ◦C for 30 s, 55 ◦C for
30 s, 72 ◦C for 90 s, and followed by 72 ◦C for 7 min. PCR prod-
ucts were cloned into the pGEM-T Easy vector (Promega), and
the sequence integrity was confirmed by sequence analysis
with the cloning primers and with two additional primers
(5ꢀ-GGT TCA CTC TGA CAG CAC TAA GG-3ꢀ and 5ꢀ-GCA GGT
GGT ACC CAG CCA AAG-3ꢀ). The sequence of the isolated
CYP2J2 cDNA completely agreed with that of the registered
CYP2J2 cDNA sequence in GenBank (accession no. U37143).
The insert cDNAs were excised from the corresponding pGEM-
T Easy vectors containing native or modified CYP2J2 cDNAs
by digestion with NdeI and HindIII. They were then purified,
and subsequently ligated into the expression vector pKSN2
at the corresponding restriction sites to construct the expres-
sion plasmids (pK2J2, pKD19, and pKD34) encoding 2J2 (wild
type), D19, and D34, respectively. E. coli JM109 was trans-
formed with pK2J2, pKD19, or pKD34 for expression experi-
ments.

steroids 7 1 ( 2 0 0 6 ) 849–856
851
phate (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol,
and 0.05% cholate) and stored at −80 ◦C. The N-terminal dele-
tion mutant CYP2J3 (dN2J3) was expressed in E. coli and puri-
fied as described previously [24]. The final preparations were
analyzed for their purity by 10% SDS-polyacrylamide gel elec-
trophoresis according to the method of Laemmli [28]. Protein
concentrations were determined by the BCA protein assay kit
(Pierce).
2.3.
Overproduction of recombinant CYP2J2 in E. coli
The E. coli cells harboring the CYP2J2 expression plasmid
were transformed with pGro7 containing the GroEL–GroES
gene, and selected with chloramphenicol (20 ␮g/ml). The dou-
bly transformed E. coli cells were precultured in LB medium
(15 ml) containing ampicillin (50 ␮g/ml) and chlorampheni-
col (20 ␮g/ml) at 37 ◦C overnight, and then 1 ml of the cul-
ture was transferred into 40 ml of TB medium containing
5 mg/ml arabinose, and maintained at 37 ◦C. Upon reaching
mid-log phase (A600 = around 0.6), the culture temperature was
reduced to 28 ◦C, and isopropyl 1-thio-␤-d-galactopyranoside
and ␦-aminolevulinic acid were added to the final concentra-
tions of 1 and 0.5 mM, respectively. The cultures were main-
tained for 20 h or 44 h at 28 ◦C. The following operations except
the measurement of P450 spectrum were carried out at 4 ◦C
or on ice. The cells were harvested by centrifugation, sus-
pended in a glycerol buffer (100 mM potassium phosphate
(pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol),
and treated with 1 mg/ml lysozyme for 1 h. After lysis, cells
were disrupted by sonication and centrifuged at 500 × g for
10 min, and the supernatant was recentrifuged at 100,000 × g
for 1 h. The supernatant and pellets were separated as cytosol
and membrane fractions, respectively. The membrane fraction
was suspended in the glycerol buffer and solubilized with 0.5%
Emulgen 911 (final concentration 0.5%) for 30 min. After cen-
trifugation at 100,000 × g for 30 min, the supernatant was col-
lected. The cytosol and solubilized membrane fractions were
subjected to the measurement of P450 contents by a reduced
CO-difference-spectrum [27].
2.5.
Assay of vitamin D 25-hydroxylase activity
Assay methods were described previously [29]. Briefly, the
purified P450 (10–150 pmol), 1.0 unit of NADPH-P450 reduc-
tase, and a substrate (25 nmol of vitamin D3, 25 nmol of vita-
min D2, or 5 nmol of 1␣(OH)D3) were mixed in 0.49 ml of
reaction buffer containing 100 mM Tris–HCl (pH 7.7 at 37 ◦C)
and 1 mM EDTA. Reaction was started by adding 10 ␮l of
50 mM ␤-NADPH. Under these conditions, the reaction prod-
ucts linearly increased with amount of P450 in range of
10–150 pmol and up to 10 min. After a 5 or 10 min incuba-
tion at 37 ◦C, reaction products were extracted with 4-ml
of benzene and subjected to HPLC analysis. The HPLC sys-
tem was composed of a PU-980 pump, a UV975 detector,
and a Fine Pack SIL-5 column (4.6 mm × 250 mm) (JASCO Co.
Ltd. Tokyo, Japan). 25(OH)D2 and 25(OH)D3 were eluted by
Solvent A (hexane:isopropanol:methanol = 93:5:2) at a con-
stant flow rate of 1.2 ml/min. 1␣,25(OH)2D3 was eluted by sol-
vent B (hexane:isopropanol:methanol = 86:7:7) at a constant
flow rate of 1.4 ml/min. The 25-hydroxylated products were
identified by comparison of retention times with authentic
25-hydroxylated compounds and quantified from the peak
heights on the normal phase HPLC. The separated products
were subjected to further identification using reverse phase
HPLC with a Fine Pack SIL C18T-5 column (4.6 mm × 250 mm).
Samples were eluted by 95% methanol at a flow rate of
1.0 ml/min for 25(OH)D2 and 25(OH)D3. Kinetic parameters
(Km and Vmax) were determined using Lineweaver–Burke plots
with the substrate concentrations of 0.625, 1.25, 2.5, and 5 ␮M.
2.4.
CYP2J3
Purification of recombinants of CYP2J2 and
All purification procedures were carried out at 4 ◦C. The cells
expressing D34 were grown in 600 ml of TB medium for 44 h at
28 ◦C, and the membrane fraction was prepared as described
above. After solubilization with sodium cholate (final concen-
tration 0.5%) and centrifugation at 100,000 × g for 30 min, the
supernatant was loaded onto an ␻-aminohexyl Sepharose col-
umn (1.6 cm × 3.0 cm) equilibrated with buffer A (10 mM potas-
sium phosphate (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 20%
glycerol, and 0.5% cholate). The column was washed with two
column volumes of buffer A, and proteins were eluted step-
wise with the same buffers containing 0.1% and 0.5% Emul-
gen 911. The eluted fractions (0.5% Emulgen) containing P450
were pooled, and dialyzed against buffer B (10 mM potassium
phosphate (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 20% glyc-
erol, and 0.3% cholate) overnight. The dialyzed fractions were
loaded onto a hydroxyapatite column (1.6 cm × 2.0 cm) equi-
librated with buffer B, and washed with more than 100 ml
of buffer B to remove Emulgen 911 completely. Proteins were
eluted stepwise with buffer B containing 100, 150, and 500 mM
potassium phosphate (pH 7.4). The fractions (500 mM eluate)
containing P450 were dialyzed against buffer B and loaded
onto a DEAE-Sepharose column (1.6 cm × 3.0 cm) equilibrated
with buffer B. The column was washed with buffer B and P450
was eluted stepwise with buffer B containing 100 and 150 mM
potassium phosphate (pH 7.4). The P450 fraction (150 mM elu-
ate) was dialyzed against buffer C (10 mM potassium phos-
2.6.
Mass spectrometric analysis of the metabolites
Metabolites were purified by the normal and reverse phase
HPLC. The isolated products were dissolved in ethanol (50 ␮l)
and infused via a syringe pump (Holliston, MA, USA) at
10 ␮l/min into the ionspray source of an Applied Biosys-
tems/QSTAR XL (Concord, Ont., Canada) triple quadrupole
instrument. The collision gas was nitrogen and the instrument
was optimized for maximum response at unit mass resolution.
The data were averaged over 100 scans collected in duplicate.
3.
Results
3.1.
coli
Heterologous expression of human CYP2J2 in E.
We have prepared three CYP2J2 expression vectors, for a full-
length protein (2J2) as well as for two N-terminal deletion
mutant proteins (D19 and D34), as shown in Fig. 1, since
substitutions and/or deletions of the N-terminal hydrophobic

852
steroids 7 1 ( 2 0 0 6 ) 849–856
Table 1 – Comparison of expression levels of CYP2J2
mutants and their distribution in E. coli
Expression
vector
Amount of P450 expression
(nmol/l of culture)
Fig. 1 – N-Terminal region of deletion mutants of CYP2J2.
2J2 indicates the wild type with a modification at the
second amino acid residue (Lue to Ala). D19 and D34
represent deletion mutants of the anchor region lacking
2-19 and 2-34 residues, respectively. A putative membrane
anchor region is underlined in 2J2. Substitution of amino
acids and the corresponding codons are as follows: for D19,
19R(CGC) to M(ATG), 20T(ACT) to A(GCT), 21L(CTC) to L(CTG),
22L(CTA) to L(TTA), 23L(CTG) to L(TTA), and 24G(GGC) to
A(GCA), and for D34, 34F(TTT) to M(ATG), 35L(CTC) to
A(GCT), 37R(AGA) to R(CGT), and 38R(CGG) to R(CGT). The
substituted amino acids are indicated in bold letters in D19
and D34.
20 h
44 h
Cytosol Membrane Cytosol Membrane
a
pK2J2/pGro7
pKD19/pGro7
pKD34/pGro7
–
–
0.4
–
–
128
–
–
145
1.0
138
182
a
Not detected.
localized in the membrane fraction. Long culture times (44 h)
did not increase the P450 level (Table 1).
3.2.
Purification of D34
D34 was purified from the membrane fraction because of
its higher P450 content therein relative to the cytosol frac-
tion (Table 1). The membrane fraction was solubilized and
applied to hydrophobic and subsequent hydroxyapatite col-
umn chromatographies. In the hydroxyapatite column step,
Emulgen 911 was completely removed by extensive washing
(while monitoring its UV absorption at 275 nm), since it signif-
icantly inhibits the vitamin D hydroxylation activity of CYP2J2
(data not shown). Finally, samples were subjected to a DEAE-
Sepharose column for purification and concentration, and dia-
lyzed against a buffer (containing 0.05% cholate) that did not
inhibit vitamin D 25-hydroxylase activity. SDS-polyacrylamide
gel electrophoresis of the final preparations showed single
major bands with the predicted molecular weights (Fig. 3). The
P450 contents of D34 and dN2J3 (recombinant CYP2J3) were
more than 10 nmol P450/mg proteins.
region (membrane anchor region) of microsomal P450 improve
expression in E. coli [30]. 2J2 has one Leu (CTC) to Ala (GCT) sub-
stitution at the second codon, as is also the case for CYP17
[31]. D19 lacks half of a putative membrane anchor region,
and the N-terminal sequence is modified similarly to that of
a CYP3A4 mutant [32]. D34 was completely devoid of the N-
terminal hydrophobic region, with the new N-terminal region
modified like that of the CYP2J3 mutant [24]. In spite of these
modifications, none of the constructs expressed P450 when
analyzed by CO-difference spectra. Since CYP2J3 expression
was markedly improved by the co-expression of E. coli chap-
erone proteins [24], we applied the same strategy to CYP2J2
expression experiments. Co-expression of GroEL–GroES dra-
matically increased the expression levels of the P450 (Fig. 2
and Table 1). The P450 expression level of E. coli cells containing
pKD34/pGro7 was more than 100 times higher than of those
containing pKD19/pGro7. D34 was distributed both in cytosol
and membrane fractions equally, whereas D19 predominantly
3.3.
Analysis of 25-hydroxylation activities of CYP2J
enzymes
vitamin D 25-hydroxylation assays were carried out using a
reconstituted system consisting of NADPH-P450 reductase.
The products were analyzed by comparison of their reten-
tion times with authentic 25-hydroxylated compounds, as
described in Section 2. Fig. 4 shows the typical results of the
HPLC analysis for 1␣(OH)D3, vitamin D3, and vitamin D2 on the
normal phase HPLC. The major peaks were identified as 25-
hydroxylation products. Several minor peaks of unidentified
products were also observed. The 25-hydroxylation products
were collected and their identities confirmed by reverse phase
HPLC (data not shown). The product identified as 25(OH)D2 by
HPLC was subjected to mass spectrometric analysis. As shown
in Fig. 5, the major molecular ion (m/z 413, M + H+) clearly
indicates the metabolite was the monohydroxylated product
of vitamin D2. The amounts of the products were calculated
from their peak heights, and kinetic parameters were deter-
mined as described in Section 2 (Table 2). Interestingly, human
CYP2J2 exhibited higher activity toward vitamin D2 than D3
(0.16 and 0.087 nmol/min/nmol P450, respectively), whereas
rat CYP2J3 showed higher activity toward vitamin D3 than
D2 (1.4 and 0.86 nmol/min/nmol P450, respectively) (Table 3).
Fig. 2 – Reduced CO-difference spectrum of recombinant
D34 expressed in E. coli. D34 was co-expressed with
GroES–GroEL and purified from a membrane fraction. The
analysis was performed with a membrane fraction (2.4 mg)
solubilized with 0.5% Emulgen 911 in 100 mM potassium
phosphate (pH 7.4), 1 mM EDTA, and 20% glycerol.

steroids 7 1 ( 2 0 0 6 ) 849–856
853
metabolized to pharmacologically active metabolite via the
hydroxylation catalyzed by CYP2J2 or CYP2J3 in the intestine
[33]. The activity of CYP2J2 and CYP2J3 toward vitamin D was
lower compared to that toward ebastin (Table 3).
4.
Discussion
We have analyzed the vitamin D 25-hydroxylation activity of
human CYP2J2 and compared it with that of the rat’s orthol-
ogous P450, CYP2J3, since CYP2J3 shows markedly high 25-
hydroxylation activity toward vitamin D3, and appears to play
an important role as a vitamin D 25-hydroxylase in the rat’s
liver [24]. Here, we have clearly demonstrated that human
CYP2J2 also exhibits significant 25-hydroxylation activity for
vitamin D3, even though human CYP2J2 and rat CYP2J3 show
a 27% amino acid sequence difference. Interestingly, human
CYP2J2 exhibited two-fold higher hydroxylation activity for
vitamin D2 than for vitamin D3. This preference was oppo-
site to that of the rat CYP2J3 (Table 3). Therefore, human and
rat CYP2J proteins exhibit distinct preferences for vitamin D
compounds.
CYP2J2 has a high catalytic ability for drugs and xenobiotics
such as ebastin [33], in keeping with the present observa-
tion that CYP2J2 prefers vitamin D2 (exogenous) to vitamin D3
(endogenous). However, when the same amount of vitamins
D2 and D3 is orally administered, rats exhibits a higher blood-
concentration of 25(OH)D2, whereas, humans show a higher
concentration of 25(OH)D3 [6,34,35]. These results are opposite
to those one would predict from the 25-hydroxylation prefer-
ences of CYP2J2 and CYP2J3. Therefore, the serum 25(OH)D
status is likely to be determined by several factors, such as
the substrate specificity of other 25-hydroxylases, affinity for
vitamin D binding protein, clearance rates, and absorption
efficiencies in the intestine.
Fig. 3 – SDS-PAGE analysis of the purified CYP2J2 (D34) and
CYP2J3 (dN2J3) proteins used for activity analysis. Lane 1:
molecular weight markers; lane 2: CYP2J2 (300 ng); lane 3:
CYP2J3 (300 ng). Gel were visualized by CBB staining.
Calculated molecular weights from amino acid sequences
are 54202 and 54373 for D34 and d2J3, respectively.
Humans express two microsomal P450’s (CYP2R1 [17] and
CYP3A4 [19]) and a mitochondrial P450 (CYP27A1 [13]) that
exhibit vitamin D 25-hydroxylation activity. CYP2R1 is mainly
expressed in the liver and testis, and hydroxylates both vita-
min D3 and vitamin D2 at C-25 with equal efficiency. It is the
most important enzyme for 25(OH)D3 synthesis in humans,
CYP2J2 showed no 1␣-, 24-, and 26-hydroxylation activity
toward 25(OH)D3, and no 2␣- and 16␣-hydroxylation activ-
ity toward testosterone, indicating similar enzymatic char-
acteristics to CYP2J3 (data not shown). Ebastin, a histamine
H1-receptor antagonist, is a well-characterized drug that is
Fig. 4 – Normal phase HPLC analysis of the metabolites of 1␣(OH)D3, vitamin D3, and vitamin D2 by purified D34. (A)
1␣(OH)D3; (B) vitamin D3; (C) vitamin D2. Elution positions of authentic 1,25-(OH)2D3, 25(OH)D3, and 25(OH)D2 are indicated
by arrows. Detailed HPLC conditions are described in Section 2.

854
steroids 7 1 ( 2 0 0 6 ) 849–856
Fig. 5 – Mass spectrum of the 25-hydroxylated metabolite of vitamin D2 in Fig. 4(C). The purified metabolite was analyzed
by mass spectrometry as described in Section 2. The peak of m/z 413 agrees with the molecular ion of 25(OH)D2 + H+.
Table 2 – Kinetic parameters of CYP2J2 (D34) for Vitamin D compounds
Substrates
Hydroxylation
position
Turnover number
(nmol/min/nmol P450)
Km (␮M)
Vitamin D3
Vitamin D2
1␣(OH)D3
25(OH)D3
C-25
C-25
C-25
C-24 or 1␣
0.087 0.013a
0.16 0.03
2.2 1.0
7.7 1.2a
2.0 0.3
4.4 0.7
n.d.
n.d.b
a
The results are means S.E. of at least three separate experiments.
n.d., not detected.
b
since the patient carrying a mutation in the CYP2R1 gene
shows very low 25(OH)D level in the blood [18]. Cheng et al.
confirmed that the single point mutation of CYP2R1 protein
abolishes 25-hydroxylation activity toward vitamin D3 and
strongly diminishes 25-hydroxylation activity toward vitamin
D2 on the expression experiments of CYP2R1 cDNA using HEK
293 cells [18].
CYP3A4, a major P450 involved in xenobiotics metabolism
in the liver and intestine, shows 25-hydroxylation activity for
vitamin D2 but not for vitamin D3 [19]. CYP27A1 is expressed
ubiquitously [36,37], and exhibits 25-hydroxylation activity
toward vitamin D3 and 24-hydroxylation activity toward vita-
min D2 [13]. Our results have demonstrated that CYP2J2
exhibits a relatively higher 25-hydroxylation activity for vita-
min D2 than for vitamin D3. It localizes in the intestine, liver,
and heart [38]. Therefore, it is likely that, similar to xeno-
biotics, vitamin D2 undergoes a first-pass activation in the
intestine by CYP2J2 or CYP3A4 [39], because vitamin D2 is sup-
plied only through the diet. The physiological importance of
CYP2J2 as a vitamin D3 25-hydroxylase in vivo is unclear since
its turnover numbers are low than that for vitamin D2 and
its apparent Km values appear to be higher than the concen-
tration of circulating vitamin D3 (ca. 2.5–70 nM for humans).
Taken together, humans have at least four vitamin D 25-
hydroxylases that show distinct features, i.e. substrate speci-
ficity, position selectivity, and tissue distribution.
Although CYP2J2 expressed in baculovirus and yeast sys-
tems has been used to analyze its catalytic properties [33,38],
no expression experiment of CYP2J2 in E. coli has been
reported. Our E. coli expression system has achieved a very
high expression level of CYP2J2 by combining the deletion of
a putative membrane anchor region in the N-terminal with
the co-expression of the E. coli chaperone protein GroEL–GroES
(Table 1). Therefore, the modification applied here is useful
for the preparation of CYP2J2 protein. It should be noted that
the truncations of the N-terminal hydrophobic region do not
severely affect the catalytic activity and substrate specificity of
P450 [24,40]. The deletion of an N-terminal hydrophobic region
may account for the equal distribution of D34 in cytosol and
membrane fractions (Table 1).
Table 3 – Comparison of catalytic activities of CYP2J2 and
CYP2J3
Substrate
Turnover number
(nmol/min/nmol P450)
CYP2J2
CYP2J3
Vitamin D3
Vitamin D2
1␣(OH)D3
Ebastina
0.087
0.16
2.2
40.6b
1.4
0.86
22
6.4b
Recently, Shinkyo et al. analyzed the 25-hydroxylation
activity of CYP2J2 toward vitamin D using baculovirus-infected
insect cells [41]. The reported turnover numbers for 1␣(OH)D3,
a
A potent and selective histamine H1-recepter antagonist in vivo.
Ref. [33].
b

steroids 7 1 ( 2 0 0 6 ) 849–856
855
[11] Ohyama Y, Yamasaki T. Eight cytochrome P450s catalyze
vitamin D2, and 1␣(OH)D2 were 0.060 0.006, 0.020 0.003,
and 0.860 0.003 min−1, respectively. Their values for vitamin
D2 and 1␣(OH)D3 are much lower than found here (about 8
and 35 times, respectively), and moreover, no activity toward
vitamin D3 was found in their study. However, the prefer-
ence of vitamin D2 structure to that of D3 can be seen in
both their results (1␣(OH)D3 and 1␣(OH)D2) and our results
(vitamins D3 and D2). The differences between the two stud-
ies is likely to be due to the differences in the samples,
i.e. crude CYP2J2 in the microsomes of insect cells versus
purified CYP2J2 protein. Another reason may be the poly-
morphisms of the CYP2J2 gene, of which there are five vari-
ants, according to King et al. [42]. These CYP2J2 variants
showed different hydroxylation activity toward arachidonic
acid [42].
In conclusion, CYP2J2 is the fourth P450 exhibiting vita-
min D3 25-hydroxylation activity in humans. It catalyzes the
25-hydroxylation of vitamins D3 and D2, (D3 < D2), although
CYP2J2 appears not to be a principal vitamin D 25-hydroxylase
in humans compared to CYP2J3 in rats. Future research will be
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