From the Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Box 578, Uppsala S-751 23, Sweden
Received for publication, July 24, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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A cytochrome P450 expressed in pig liver was
cloned by polymerase chain reaction using oligonucleotide primers based
on amino acid sequences of the purified taurochenodeoxycholic acid
6 The formation of bile acids from cholesterol in the liver involves
a series of cytochrome P450-dependent hydroxylations of the
steroid nucleus as well as the side chain. The two primary bile acids
formed in most mammals are 3 In previous reports from this laboratory, the purification and
characterization of the taurochenodeoxycholic acid 6 In the present study, we report on the cDNA cloning and expression
of a taurochenodeoxycholic acid 6 Materials--
Livers and other tissues from castrated,
otherwise untreated, male pigs were obtained from the local
slaughterhouse. RNeasy Midi kits, QIAprep Spin Miniprep kit, and
Plasmid Maxi kit were from Qiagen. Restriction enzymes, SureClone
Ligation kit, and ECL Western blotting analysis system were from
Amersham Pharmacia Biotech. pBluescript II KS was purchased from
Stratagene. Oligonucleotides were obtained from Life Technologies and
Interactiva. Taq polymerase and oligo(dNTP) mix were from PE
Applied Biosystems and Advanced Biotechnologies. The reverse
transcription system was from Promega, and the 1.5-kb DNA ladder and
5'-rapid amplification of cDNA ends (RACE) system were from Life
Technologies, Inc. T4 DNA ligase and buffer were from New England
Biolabs. Horseradish peroxidase-conjugated goat anti-rabbit IgG
antibodies were purchased from Bio-Rad. The megaprime DNA labeling
system, CYP IVA ECL Western blotting kit, and
[1-14C]lauric acid (53 Ci/mol) were from Amersham
Pharmacia Biotech. Tauro[24-14C]chenodeoxycholic acid
(0.8 Ci/mol) was synthesized according to Norman (6). All chemicals
used were of first grade quality.
Cloning of cDNA from Pig Liver--
Total RNA isolated from
pig liver using RNeasy Midi kits was reversed transcribed, and first
strand cDNA was used directly for PCR (RT-PCR).
Oligonucleotide primers for the initial PCR were designed from amino
acid sequences obtained from the purified taurochenodeoxycholic acid
6 Expression of cDNA in COS-1 Cells--
A cDNA sequence
for cloning into the expression vector pSVL (Amersham Pharmacia
Biotech) was obtained by RT-PCR of total RNA from pig liver using two
primers based on sequences in the 5'- and 3'-end, respectively, of the
deduced coding region. The forward primer (Xho-N,
5'-CTCTCGAGCGTGCACCATGGGGGTCCC-3') contained a restriction site for
cloning (XhoI) and a conserved eukaryotic initiation
site sequence as described by Kozak (8). The reverse primer contained
the XbaI restriction site for cloning and the most
3'-nucleotides of the deduced coding sequence (Xba-C,
5'-TTTCTAGAATGGTGTCTTCCTGAGGTTCAG-3'). The PCR cycles were according to
the procedure above. The PCR product was gel purified and cloned into a
pSVL vector. The cloned pSVL-CYP4A21 was amplified and purified using
plasmid Maxi columns (Qiagen), sequenced, and subsequently transfected
into COS-1 cells by the DEAE-dextran method as described by Esser
et al. (9) or by electroporation (settings 0.4 kV, 100 microfarads). Transfected cells were grown for 48 h at
37 °C, 5% CO2, in Dulbecco's modified Eagle's medium
supplemented with fetal calf serum (10%), glutamine, and antibiotics,
then washed and harvested. Microsomes from COS cells were isolated by
differential centrifugation according to Clark and Waterman
(10), resuspended, and homogenized in 100 mM potassium
phosphate, pH 7.4, containing 0.1 mM EDTA. Protein concentration was determined using BCA reagent (Pierce).
Activity Assay--
14C-Labeled
taurochenodeoxycholic acid (25, 145, and 200 µM) was
incubated with freshly prepared microsomes from transfected COS cells.
In addition to microsomes and substrate, the incubation mixture
contained 1 unit of NADPH-cytochrome P450 reductase from pig liver
microsomes (11), 5 µM dithiothreitol, and 2 µM NADPH in a total volume of 1 ml of 100 mM
potassium phosphate, pH 7.4, containing 0.1 mM EDTA.
Incubations were performed at 37 °C for 20 or 30 min and terminated
with 5 ml of ethanol. The incubation mixtures were prepared for thin
layer chromatography as described previously (5). The chromatoplates
were analyzed by radioactivity scanning. Two unlabeled authentic
6-hydroxylated chenodeoxycholic acid metabolites,
3
As positive controls of the activity assays, freshly prepared pig liver
microsomes were used and incubated with respective substrate according
to the procedure described above except that NADPH-cytochrome P450
reductase was omitted.
Western Blotting--
SDS-PAGE was carried out according to
Laemmli (13) with 15% acrylamide and 0.09% bisacrylamide slab gels
containing 0.1% SDS. The proteins on the gels were electrophoretically
blotted onto nitrocellulose filters. Western blot analysis was
performed using polyclonal antibodies raised against the purified
6 Northern Blot Analysis--
Total RNA from pig liver (20 µg)
was electrophoresed on 1.2% agarose gel containing formamide as
described by Sambrook et al. (14) and transferred to
Hybond-N membranes (Amersham Pharmacia Biotech). A nucleotide sequence
corresponding to the coding sequence of the cloned cDNA was
32P-labeled using a Megaprime labeling kit and used as
probe. The membrane was hybridized with the 32P-labeled
probe in hybridization buffer containing 50% deionized formamide, 5×
SSC, 100 µg/ml denatured single-stranded DNA, 100 µg/ml total RNA,
and 1× PE (50 mM Tris-HCL, pH 7.5, 0.1%
sodiumpyrophosphate, 1% SDS, 0.2% polyinylpyrolidine, 0.2% Ficoll, 5 mM EDTA, 0.2% bovine serum albumin). Hybridization was
performed overnight at 42 °C. The membrane was washed twice in each
of the following solutions, 2× SSC containing 0.1% SDS for 10 min at
room temperature, 2× SSC containing 0.1% SDS for 30 min at 62 °C,
and 0.2× SSC for 10 min at room temperature. The membrane was exposed
to Fuji RX film.
RT-PCR for Tissue Distribution Studies--
Total RNA was
extracted from nine different tissues of pig (heart, muscle, intestine,
spleen, thymus, lung, adrenal gland, kidney, and liver) using RNeasy
Midi kits or according to Chomczynski and Sacchi (15). The
quality of RNA was checked by formamide gel electrophoresis. Total RNA
(1 µg) from each tissue was reversed transcribed, and the cDNA
obtained was used for PCR. A reverse primer (RNON-CONS,
5'-TGGAGTCGTGACCTGCAGC-3'), which hybridizes to position 995 of CYP4A21
(see Fig. 1) was used together with the forward primer Xho-N described
above. PCR was performed in presence of 2.5 mM
Mg2+ and 10% dimethyl sulfoxide for 30 cycles (94 °C
for 1 min, 58 °C for 1 min, 72 °C for 2 min) and 72 °C for
10 min. The PCR products were analyzed by agarose gel electrophoresis.
Point Mutations of the CYP4A21 Sequence--
The sequence of
CYP4A21, from the restriction site AatII at position 951 to
position 1578 (Fig. 1), was amplified by PCR using CYP4A21 cDNA as
template. A forward primer,
5'-CTTGACGTCCGTGCCGAAGTGGACACGTTCATGTTCGAGGGTCATGACACCACAGCC-3' containing the restriction site AatII for cloning and
nucleotides coding for three point mutations (A314F, A315E, S319T) was
used in combination with a reverse primer, 3CB
(5'-TTTCTAGAGCTTGTCCTTGTCCCCACA-3', which hybridizes to a sequence in
the 3'-untranslated region (3'-UTR). PCR was performed in presence of
2.5 mM Mg2+ and 10% dimethyl sulfoxide for 30 cycles (94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min) and
72 °C for 10 min. The 600-bp PCR product was gel-purified, digested
with AatII and XbaI, and subcloned into a
pBluescript IIKS-ligated CYP4A21 sequence between positions 951 and
1578. The mutated sequence of CYP4A21 thus obtained was digested
further with the restriction enzymes BsmBI and
Bsu36I, and the fragment between nucleotides 588 and 1535 (Fig. 1) was subsequently cloned into the corresponding restriction
sites of the expression plasmid pSVL-CYP4A21. The mutated CYP4A21
sequence, cloned into pSVL, was verified by sequencing and used for
transfection of COS cells by the DEAE-dextran method (9).
Cloning and Nucleotide Sequencing of the Pig Liver
Taurochenodeoxycholic Acid 6
The complete nucleotide sequence of the CYP4A21 and the deduced amino
acid sequence are shown in Fig. 1.
Underlined sequences correspond to peptides obtained by
tryptic cleavage and N-terminal sequencing of the purified protein as
reported previously (5). All tryptic fragments have Arg and Lys in
positions that permit trypsin cleavage, and the peptide sequences
completely match those encoded by the cDNA. Of the 20 N-terminal
residues sequenced in the purified protein, 19 were in full agreement
with those of the deduced sequence. One residue, Ala in position 17, was replaced by Ser in the encoded sequence. The N terminus of the
deduced sequence had an additional Met in the 5'-end compared with that of the purified protein.
The cDNA is 2,400 bp long and contains a 37-nucleotide 5'-UTR, a
1,515-bp open reading frame, and an 848-bp 3'-UTR downstream from the
TAA terminal codon. The cDNA encodes a protein of 504 amino acids
containing the P450 signature motif of 10 residues (444)
which includes the invariant cysteine residue that ligates the heme
iron (16). The conserved poly(A) additional site signal (17) could not
be identified in the 3'-UTR, indicating that the (T)12
primer used adhered to a poly(A)-rich sequence upstream from the
poly(A) tail. The terminal nucleotides of the 3'-UTR are thus likely
missing in the cDNA sequence shown in Fig. 1.
The DNA sequence and the predicted amino acid sequence exhibit between
75 and 79% overall sequence identity with the reported sequences for
known members of the CYP4A subfamily from rabbit and human (18-22). It
is generally considered that all cytochrome P450 enzymes share a
largely conserved tertiary structure core of amino acids (23). A
hydrophobic sequence of about 20 residues in the N-terminal of
eukaryotic P450 enzymes is thought to represent a transmembrane anchor.
In the deduced amino acid sequence of CYP4A21 this N-terminal sequence
is shorter by six residues compared with other CYP4A subfamilies. A
unique, highly conserved region exclusively found in members of the
CYP4A and 4B subfamilies (24) is located around position 315. In
CYP4A21, however, amino acid substitutions in this region are found. As
shown in Fig. 2, Phe and Glu are replaced by
Ala in positions 314 and 315, and Thr is replaced by Ser in position
319 of CYP4A21.
The sequences in the 5'-UTR and 3'-UTR of CYP4A21 show no overall
identity with those of other members of the CYP4A subfamily. A short
sequence between nucleotides 1686 and 1721 in the 3'-UTR of CYP4A21
shows 94% identity with that of a sequence in the 3'-UTR of CYP4A11
(1716-1751). The significance of this similarity is not known at present.
Expression in COS-1 Cells--
To ascertain that the cloned
cDNA encodes a functional enzyme, a nucleotide sequence from
position 37 to 1549 (Fig. 1) was produced by PCR and inserted into the
expression vector pSVL for transfection of COS-1 cells. Mock
transfection with the pSVL vector without the cDNA insert was used
as a control. Microsomes were isolated from transfected cells by
differential centrifugation and subsequently used for the assay of
hydroxylase activity and Western blot analysis.
6
Microsomes of the cells transfected with CYP4A21 cDNA catalyzed
6
Microsomes isolated from COS cells transfected with CYP4A21 cDNA
did not show Western Blot Analysis of Microsomes from Transfected COS
Cells--
Polyclonal antibodies raised against the purified
taurochenodeoxycholic acid 6 Northern Blot Analysis--
Total RNA from pig liver was subjected
to Northern blot analysis. A nucleotide sequence corresponding to the
coding sequence of CYP4A21 was labeled and used as probe. As shown in
Fig. 4, the probe hybridized with one major
band corresponding to ~2.5 kb. The cloned cDNA is a nucleotide
sequence of 2.4 kb, but a proper poly(A) signal sequence in the
terminal 3'-UTR is missing, indicating that the mRNA is longer. An
mRNA of 2.5 kb could thus correspond to the full-length
mRNA.
RT-PCR for Tissue Distribution Studies--
Members of the CYP4A
gene subfamily have been found in various mammalian tissues including
the liver, kidney, lung, intestine, brain, prostate, uterus, and
placenta (30). Considering the high homology between CYP4A genes, an
RT-PCR procedure was chosen for investigation of tissue expression of
CYP4A21 in pig. A CYP4A21-specific reverse primer (RNON-CONS), which
hybridizes to position 995 of CYP4A21 but not to the corresponding
conserved sequence of CYP4A fatty acid hydroxylases, was used in
combination with a forward primer (Xho-N) (see "Experimental
Procedures"). As shown in Fig. 5, an
intense band corresponding to the size of the expected amplified region
(956 bp) was obtained using total RNA from liver. A faint band of the
same size was also seen with total RNA from kidney, whereas total RNA
from other tissues (heart, muscle, intestine, spleen, thymus, lung, and
adrenal gland) did not generate bands of similar size. Because of the
RT-PCR result using total RNA from pig kidney, the hydroxylase activity
toward taurochenodeoxycholic acid was tested using pig kidney
microsomes (1 mg and 5 mg). The activity was, however, below the limit
of detection. The RT-PCR and activity measurement experiments indicate
a substantially lower expression of the enzyme in kidney compared with
liver.
Expression of a Point-mutated Sequence of CYP4A21--
To study
the importance of Ala-314, Ala-315, and Ser-319 for the
6
Expression of a protein in COS cell microsomes was confirmed by Western
blot analysis using antibodies raised against rat CYP4A. An
immunoreactive band with an electrophoretic mobility identical to that
of the expressed CYP4A21 and of the purified 6 The primary structure of the taurochenodeoxycholic acid
6 An unexpected difference between the deduced amino acid sequence of
CYP4A21 and those of other hitherto cloned members of the CYP4A
subfamily is amino acid substitutions found in the otherwise conserved
sequence around residue 315 (24). Based on homology alignment (23, 32)
this part of the sequence is located in the center of the I-helix close
to the heme in the active site. Amino acids in this region have been
reported to define the steric environment of the heme group and
contribute to the preference for Besides structural considerations, the CYP4A21 is interesting
also from an evolutionary point of view. The pig is unique in having
hyocholic acid as the main trihydroxylated bile acid. The ratio between
hyocholic acid and chenodeoxycholic acid in pigs is comparable to that
of cholic acid and chenodeoxycholic acid in most other mammals. It has
been speculated that the ancestor of the domestic pig had the ability
to carry out 12 In humans, increased amounts of hyocholic acid have been found in
plasma and urine from patients with cholestatic liver disease and also
in fetal blood sample. It seems likely that hydroxylations of bile
acids in man, such as 6 In conclusion, this paper describes the cloning and deduced primary
structure of a novel CYP4A enzyme (CYP4A21) with the ability to
6-hydroxylase. This enzyme catalyzes a 6
-hydroxylation of
chenodeoxycholic acid, and the product hyocholic acid is considered to
be a primary bile acid specific for the pig. The cDNA encodes a
protein of 504 amino acids. The primary structure of the porcine
taurochenodeoxycholic acid 6
-hydroxylase, designated CYP4A21, shows
about 75% identity with known members of the CYP4A subfamily in rabbit
and man. Transfection of the cDNA for CYP4A21 into COS cells
resulted in the synthesis of an enzyme that was recognized by
antibodies raised against the purified pig liver enzyme and catalyzed
6
-hydroxylation of taurochenodeoxycholic acid. The hitherto known
CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins
and have frequently been referred to as fatty acid hydroxylases. A
change in substrate specificity from fatty acids or prostaglandins to a
steroid nucleus among CYP4A enzymes is notable. The results of
mutagenesis experiments indicate that three amino acid substitutions in
a region around position 315 which is highly conserved in all
previously known CYP4A and CYP4B enzymes could be involved in the
altered catalytic activity of CYP4A21.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,7
,12
-trihydroxy-5
-cholanoic acid (cholic acid)1 and
3
,7
-dihydroxy-5
-cholanoic acid (chenodeoxycholic acid). The
domestic pig, however, is virtually unable to synthesize cholic acid.
Instead, another trihydroxylated bile acid,
3
,6
,7
-trihydroxy-5
-cholanoic acid (hyocholic acid), is
formed in amounts equal to that of cholic acid in other mammals.
Formation of hyocholic acid is accomplished by a 6
-hydroxylation of
chenodeoxycholic acid or its conjugates. Hyocholic acid is considered
to be a species-specific primary bile acid in the pig. It has been
speculated that the ability to synthesize hyocholic acid in pigs
evolved from a need of trihydroxylated bile acids caused by changes in
dietary circumstances. 6
-Hydroxylation of chenodeoxycholic acid
seems to have been biochemically preferred during evolution compared
with the introduction of cholic acid biosynthesis (1). In man, small
amounts of hyocholic acid have been found in plasma and urine
especially from patients with cholestatic liver disease (2) and also in
fetal blood sample (3), indicating a different role for this bile acid
in man.
-hydroxylase from pig liver have been described (4, 5). The enzyme was shown to be a
microsomal cytochrome P450 heme protein. The N-terminal amino acid
sequence and sequences of peptides obtained after partial tryptic
digestion suggested that this enzyme belong to the cytochrome P450 4A
(CYP4A) subfamily.
-hydroxylase from pig liver. The
results confirm and extend the previous observation that this enzyme is
a, so far uncharacterized, member of the CYP4A subfamily. The novel
CYP4A enzyme was designated
CYP4A21.2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase (5). The forward primer was N2
(5'-CGTCCCGGCCCTGGCCAGCGT-3'), and the reverse primer was R-FEL
(5'-TGGGATCTGGCGCCAGCTCGAA-3'). The PCR was performed in presence of
2.5 mM Mg2+ and 10% dimethyl sulfoxide. The
cycles were as follows: 94 °C for 2 min, 55 °C for 1 min,
72 °C for 2 min for 30 cycles, and 72 °C for 10 min. The 1,430-bp
PCR product was gel purified and cloned into pUC18 using a SureClone
ligation kit and subsequently transformed in Escherichia
coli DH5
cells. Plasmid DNA was purified using DNA
minipreparation columns (Qiagen) according to the manufacturer's instruction. A plasmid containing the insert was sequenced using Dye-labeled Terminator for the sequence reaction and a ABI377 DNA
sequencer for analysis. The full-length nucleotide sequence was
obtained by 5'- and 3'-RACE using a 5'-RACE system according to the
manufacturer's instructions and a 3'-RACE procedure according to
Frohman (7). Gene-specific primers (5'-GSP1 and 5'-GSP2) used for the
5'-RACE were 5'-GSP1 (5'-TAGATGATGTCGTTCTCGAGAA-3') and 5'-GSP2
(5'-CATGCGCCGGTGCTGGAACCA-3'). For the 3'-RACE, two gene-specific
primers (3'-GSP1 and 3'-GSP2) and a (T)12 primer were used. The sequences of the gene-specific 3'-primers were: 3'-GSP1,
5'-TTCTCGAGAACGACATCATCTA-3', and 3'-GSP2,
5'-TCTGCTCGCCACAGCCATGCTTT-3'. The PCR products of the 5'-RACE and
3'-RACE, respectively, were gel purified and cloned into pUC18 and
subsequently sequenced according to the procedure above. The nucleotide
sequence of the cDNA was determined by alignment of overlapping
sequences of the 1,430-bp PCR product and the sequences of the 5'-RACE
and 3'-RACE.
,6
,7
-trihydroxy-5
-cholanoic acid and
3
,6
,7
-trihydroxy-5
-cholanoic acid (Steraloids, Inc.), were
used as external standards in thin layer chromatography. Hydroxylase
activity toward lauric acid was assayed by incubation of microsomes
from transfected COS cells with 14C-labeled lauric acid
(100 µM), 1 unit of NADPH-cytochrome P450 reductase, and
2 µM NADPH in a total volume of 0.2 ml of 0.1 M Tris-HCl, pH 7.5. Incubations were performed at 37 °C
for 20 min and terminated by the addition of 0.1 ml of 6% acetic acid
in ethanol. Samples were centrifuged and the supernatant analyzed by
high performance liquid chromatography according to Okita
et al. (12) using a Radiomatic detector.
-hydroxylase (5) as primary antibodies and horseradish
peroxidase-conjugated goat anti-rabbit IgG as secondary antibodies
(Bio-Rad). The immunoreactive protein bands were visualized with an ECL
Western blotting analysis system. The filter was stripped of bound
antibodies and immunodetected a second time with antibodies raised
against rat CYP4A (Amersham Pharmacia Biotech) according to the
manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Hydroxylase
(CYP4A21)--
Oligonucleotide primers for PCR were designed from
peptide sequences of the purified protein (5). A 1,430-bp PCR product was produced with a forward primer based on residues in the N-terminal sequence of the purified protein and a reverse primer based on a
tryptic fragment, which in an alignment with rabbit CYP4A7, was the
most C-terminal sequence obtained from the protein. The DNA sequence of
this PCR product was analyzed. Nucleotides in the 5'- and 3'-regions of
the cDNA were obtained by use of 5'- and 3'-RACE procedures. The
sequence of the full-length cDNA was finally determined by
alignment of overlapping sequences.
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Fig. 1.
cDNA and deduced amino acid sequence of
taurochenodeoxycholic acid 6 -hydroxylase from
pig liver. Underlined amino acid sequences correspond
to the N-terminal sequence and sequences of peptides obtained
previously by tryptic digestion of the purified enzyme.
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Fig. 2.
Alignment of the deduced sequences between
positions 308 and 320 of CYP4A21 and corresponding residues in the
conserved sequence common to CYP4A and CYP4B subfamilies. Residues
discussed in the paper are underlined in the CYP4A21
sequence.
-Hydroxylase activity toward taurochenodeoxycholic acid was assayed
by incubating microsomes isolated from transfected COS cells with
14C-labeled taurochenodeoxycholic acid. The conjugated
substrate was chosen because previous studies with pig liver microsomes have shown that the rate of 6
-hydroxylation was five times more efficient when taurochenodeoxycholic acid was used as substrate compared with chenodeoxycholic acid (4). The incubation mixtures were
subsequently analyzed by thin layer chromatography together with
standards of the unlabeled authentic 6
- and 6
-hydroxylated metabolites, 3
,6
,7
-trihydroxy-5
-cholanoic acid and
3
,6
,7
-trihydroxy-5
-cholanoic acid. A single radioactive
peak corresponding to the migration of the 6
-hydroxylated product
(Rf = 0.22) was seen in samples incubated with
microsomes from COS cells transfected with the pSVL-vector containing
CYP4A21 cDNA but not in samples incubated with microsomes from the
mock-transfected COS cells. The 6
-hydroxylated metabolite standard
migrated with a lower Rf value
(Rf = 0.16).
-hydroxylation of taurochenodeoxycholic acid at a rate between 5 and 50 pmol/mg of microsomal protein/min, using three concentrations of
substrate (25, 145, and 200 µM). A similar range of
activity, dependent on the substrate concentration, was found in
preparations of pig liver microsomes using the same incubation
procedure (Table I). A
Km of 62.9 µM has been calculated
previously for the purified pig liver enzyme (5). The higher rate of
6
-hydroxylation by recombinantly expressed CYP4A21 with higher
concentrations of substrate is consistent with the
Km-value for the purified enzyme and the high
concentrations of bile acids in portal venous blood (50-170
µM) (25).
Hydroxylase activities toward taurochenodeoxycholic acid and lauric
acid using microsomes from transfected COS cells and pig liver
microsomes
-hydroxylase activity toward
taurochenodeoxycholic acid in the transfected COS cell microsomes is
comparable to that in pig liver microsomes, whereas there is a marked
difference in
- and (
-1)-hydroxylation of lauric acid.
- or (
-1)-hydroxylase activity toward lauric acid.
In Table I, the hydroxylase activities toward taurochenodeoxycholic acid and lauric acid, using the same preparation of microsomes from
CYP4A21-transfected COS cells, are shown together with results using
freshly prepared pig liver microsomes. The 6
-hydroxylase activity in
microsomes from transfected COS cells and pig liver microsomes is
comparable, but the
- and (
-1)-hydroxylase activities, present in
pig liver microsomes, were not detectable with microsomes from the
transfected COS cells. It has been shown in rat and human liver that
formation of a
-hydroxy metabolite of lauric acid is a marker of
CYP4A enzyme activity, whereas other cytochrome P450 isoenzymes, like
members of the CYP2 family, hydroxylate primarily the (
-1)-position
of lauric acid (26-29). The present results strongly indicate that
6
-hydroxylation of taurochenodeoxycholic acid and
-hydroxylation
of lauric acid in pig liver microsomes are performed by two distinct
isozymes. Thus, the cloned microsomal porcine enzyme is not a pig
equivalent to the CYP4A fatty acid hydroxylase enzymes found in other species.
-hydroxylase (5) and antibodies raised
against rat CYP4A were used to detect immunoreactive proteins in
microsomes of transfected COS cells. Samples from pig liver microsomes
and the purified 6
-hydroxylase enzyme were used as positive
controls. As shown in Fig. 3, microsomal
protein from COS cells transfected with the pSVL vector containing
CYP4A21 cDNA was detected by both antibodies. The electrophoretic
mobility of the immunoreactive protein was the same as that of the
protein recognized by antibodies in pig liver microsomes and purified
protein. Microsomes from mock-transfected cells did not show
immunoreactive proteins with similar electrophoretic mobility. The
ability of the antibodies raised against rat CYP4A to bind expressed
CYP4A21 indicates that similar epitopes are recognized in both
proteins. The overall amino acid sequence identity between pig
taurochenodeoxycholic acid 6
-hydroxylase and rat CYP4A is about
68%.
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Fig. 3.
Western blot experiment with microsomes from
transfected COS cells. Microsomes from transfected COS cells and
pig liver as well as purified 6 -hydroxylase from pig liver
microsomes were subjected to SDS-polyacrylamide gel electrophoresis.
Proteins were transferred from the gel to a sheet of nitrocellulose and
incubated with polyclonal antibodies raised against the purified
protein (panel A). The filter was stripped of bound
antibodies and incubated a second time with antibodies against rat
CYP4A (panel B). Lane 1, 0.4 µg of purified pig
liver microsomal taurochenodeoxycholic acid 6
-hydroxylase;
lane 2, 60 µg of microsomes from COS cells transfected
with taurochenodeoxycholic acid 6
-hydroxylase cDNA; lane
3, 60 µg of microsomes from COS cells transfected with pSVL
vector without insert; lane 4, 40 µg of pig liver
microsomes. Immunoreactive bands are seen in lane 2 with
both antibodies (A and B) but not in lane
3. The electrophoretic mobility of the immunoreactive protein in
lane 2 corresponds to the protein detected by both
antibodies in samples of pig liver microsomes and purified
enzyme.
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Fig. 4.
Northern blot analysis of total RNA (20 µg) isolated from pig liver. A nucleotide
sequence corresponding to the coding sequence of CYP4A21 from pig liver
was labeled with 32P and used as probe according to the
hybridization procedure described under "Experimental Procedures."
The positions of ribosomal RNA 28 S (4.8 kb) and 18 S (1.9 kb) used
as built-in molecular weight markers and the estimated size of the
major band detected by the probe are indicated. This band of ~2.5 kb
probably corresponds to the full-length mRNA of
taurochenodeoxycholic acid 6 -hydroxylase from pig liver.
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Fig. 5.
RT-PCR of total RNA for tissue distribution
studies. Total RNA from different tissues were reversed
transcribed, and the first strand cDNAs were used for PCR. A
CYP4A21-specific reverse primer based on the differences between
CYP4A21 and CYP4A/4B in the sequence around residue 315 was used
together with a forward primer (Xho-N) (see "Experimental
Procedures"). The PCR products were analyzed by agarose
gel-electrophoresis using a 1-kb DNA ladder for size determination. The
samples were as follows: lane 1, heart; lane 2,
muscle; lane 3, intestine; lane 4, spleen;
lane 5, thymus; lane 6, lung; lane 7,
adrenal gland; lane 8, kidney; and lane 9,
liver.
-hydroxylase activity, a mutated sequence of the CYP4A21 was
produced using a primer containing nucleotides coding for the
corresponding conserved amino acids Phe-314, Glu-315, and Thr-319 (Fig.
2). The three mutations thus introduced in CYP4A21 were A314F, A315E,
and S319T. The mutated sequence was cloned into pSVL vector and used
for transfection of COS cells. Microsomes prepared from the transfected
COS cells were used for Western blot and activity assays.
-hydroxylase was
detected (results not shown). Microsomes from COS cells transfected
with the mutant CYP4A21 did not show hydroxylase activity toward
taurochenodeoxycholic acid or lauric acid. This indicates that the
mutated amino acids in the active site of CYP4A21 are involved in the
catalytic activity toward taurochenodeoxycholic acid but are not the
sole determinants for the CYP4A enzyme activity toward lauric acid.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase (CYP4A21) from pig liver described in this paper
reveals it to be a member of the CYP4A subfamily. The hitherto known
CYP4A enzymes catalyze
- and (
-1)-hydroxylations of fatty acids
and/or prostaglandins and are often referred to as fatty acid
hydroxylases. Despite the overall sequence similarity (74% identity)
between the human fatty acid hydroxylase CYP4A11 and the present
CYP4A21, the former showed no 6
-hydroxylase activity toward
taurochenodeoxycholic acid (31) and the latter no hydroxylase activity
toward lauric acid. A change in substrate specificity from a fatty acid
to a steroid nucleus among CYP4A enzymes is notable and is probably caused by amino acid substitutions in regions involved in substrate binding and catalytic activity.
-hydroxylation of fatty acids, a
unique feature of CYP4A fatty hydroxylases (33). The expressed mutated
CYP4A21 (A314F/A315E/S319T) did not show 6
-hydroxylase activity
toward taurochenodeoxycholic acid. Thus, introduction of the conserved
amino acids in CYP4A21 abolishes the 6
-hydroxylase activity,
indicating that Ala-314, Ala-315, and Ser-319 in the active site of
CYP4A21 are important for the catalytic activity. Introduction of these
amino acids did, however, not convert CYP4A21 into a fatty acid
hydroxylase. The presence of a CYP4A fatty acid hydroxylase in pig
liver was shown by the ability of pig liver microsomes to
-hydroxylate lauric acid. It seems likely that the substrate
specificity of CYP4A21 and CYP4A fatty acid hydroxylase(s),
respectively, in pig liver are highly restricted and determined by
residues also outside the active site. Substrate recognition sites
distal to the heme moiety have been shown to influence the catalytic
activity of rat and human CYP4As in studies using mutated enzymes
(34-36) or lauric acid analogs (37). A number of substrate-binding
residues have also been identified in the substrate access channel in a structural model of CYP4A11 (38). Those studies were, however, confined
to the issue of the regiospecificity of fatty acid hydroxylation and
catalytic activity toward different fatty acids. The results with
CYP4A21 presented here provide new information, which should be useful
in further studies on the molecular basis for CYP4A enzymes to bind and
hydroxylate a steroid nucleus.
-hydroxylation and to form cholic acid as the related
warthog now does, but this ability was subsequently lost. Formation of
hyocholic acid by 6
-hydroxylation of chenodeoxycholic acid might
have evolved as a more biochemically expedient way to fill the
requirement of trihydroxylated bile acids brought about by some change
in dietary circumstances (1). The CYP4 family is one of the oldest
cytochrome P450 families having diverged from a common ancestor over
1.25 billion years ago (30, 39, 40). Considering the speculation that
the ability to form hyocholic acid in pigs evolved late by dietary
changes, the CYP4A21 is probably a more recent cytochrome P450 compared
with the known fatty acid-hydroxylating members of the CYP4A subfamily.
In this context, it is interesting to note that the key enzyme in
formation of cholic acid, the sterol 12
-hydroxylase (CYP8B), shows a
high degree of sequence identity with the prostacyclin synthase
(CYP8A). In that case, it has been suggested that CYP8A might have
diverged from an ancient CYP8B and subsequently acquired the novel
function through extensive alteration (41).
-hydroxylation, are a way for detoxification
of accumulating cytotoxic bile acids when the normal pathways for
excretion are either disturbed (as in cholestasis) or poorly developed
(as in the fetus). CYP3A4 has been shown to carry out
6
-hydroxylation of taurochenodeoxycholic acid in experiments with
human liver microsomes and recombinantly overexpressed enzyme (31). A
role for CYP3A4 in hyocholic acid formation in man is supported by the
observation that 6
-hydroxylation of bile acids in humans is
stimulated by rifampicin, a well known inducer of CYP3A4 (42). It is
not known at present whether an additional 6
-hydroxylating enzyme,
orthologous to the porcine CYP4A21, is expressed in human or in other
species besides pig. The present results showing a low expression of
CYP4A21 in pig kidney indicate that this enzyme has functions in
addition to participating in bile acid biosynthesis in pig liver.
-hydroxylate taurochenodeoxycholic acid. In addition to its biological role in formation of hyocholic acid in the pig, this enzyme
is also interesting from evolutionary and structural aspects. The
porcine CYP4A21 opens up possibilities to study the evolutionary relationship between this enzyme and other members of the CYP4A subfamily and should contribute to our understanding of features important for the substrate specificity of CYP4A enzymes.
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FOOTNOTES |
---|
* This work was supported by Swedish Medical Research Council Project 03X-218.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ278474 SSC278474.
To whom correspondence should be addressed. Fax:
46-18-558-778; E-mail: Kerstin.Lundell@farmbio.uu.se.
Published, JBC Papers in Press, December 11, 2000 DOI 10.1074/jbc.M006584200
2
The sequence reported for the porcine
taurochenodeoxycholic acid 6-hydroxylase was designated CYP4A21 by
the P450 nomenclature committee.
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ABBREVIATIONS |
---|
The abbreviations used are:
cholic acid, 3,7
,12
-trihydroxy-5
-cholanoic acid;
chenodeoxycholic acid, 3
,7
-dihydroxy-5
-cholanoic acid;
CYP, cytochrome P450;
kb, kilobase pair(s);
RACE, rapid amplification of
cDNA ends;
RT, reverse transcription;
PCR, polymerase chain
reaction;
bp, base pair(s);
GSP, gene-specific primer;
UTR, untranslated region.
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