Cytochrome P450 Cyp4x1 is a major P450 protein
in mouse brain
Mohammed Al-Anizy
1
, Neill J. Horley
1
, C W. S. Kuo
1
, Lorna C. Gillett
2
, Charles A. Laughton
2
,
David Kendall
3
, David A. Barrett
2
, Terry Parker
3
and David R. Bell
1
1 School of Biology, University of Nottingham, UK
2 School of Pharmacy, University of Nottingham, UK
3 School of Biomedical Sciences, University of Nottingham, UK
Cytochromes P450 are a superfamily of proteins [1]
which are involved in the oxidative metabolism of both
foreign and endogenous compounds [2]. The cyto-
chrome P450 4A family is known to be highly induced
by peroxisome proliferators in mouse liver [3,4],
although there is constitutive expression of one gene
[5]. The CYP4A [6], CYP4B [7,8] and CYP4F [9,10]

E-mail:
(Received 16 November 2005, revised 20
December 2005, accepted 23 December
2005)
doi:10.1111/j.1742-4658.2006.05119.x
A novel cytochrome P450, CYP4x1, was identified in EST databases on
the basis of similarity to a conserved region in the C-helix of the CYP4A
family. The human and mouse CYP4x1 cDNAs were cloned and found
to encode putative cytochrome P450 proteins. Molecular modelling of
CYP4x1 predicted an unusual substrate binding channel for the CYP4 fam-
ily. Expression of human CYP4x1 was detected in brain by EST analysis,
and in aorta by northern blotting. The mouse cDNA was used to demon-
strate that the Cyp4x RNA was expressed principally in brain, and at much
lower levels in liver; hepatic levels of the Cyp4x1 RNA were not affected
by treatment with the inducing agents phenobarbital, dioxin, dexametha-
sone or ciprofibrate, nor were the levels affected in PPARa– ⁄ – mice. A
specific antibody for Cyp4x1 was developed, and shown to detect Cyp4x1
in brain; quantitation of the Cyp4x1 protein in brain demonstrated  10 ng
of Cyp4x1 proteinÆmg
)1
microsomal protein, showing that Cyp4x1 is a
major brain P450. Immunohistochemical localization of the Cyp4x1 protein
in brain showed specific staining of neurons, choroids epithelial cells and
vascular endothelial cells. These data suggest an important role for Cyp4x1
in the brain.
Abbreviations
DAB, 3,3¢-diaminobenzidine; TCDD, 2,3,7,8 tetrachlorodibenzo-p-dioxin.
936 FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS
Results
Identification of human CYP4x1

The predicted CYP4x1 protein has characteristics of
a functional P450, including conservation of the haem-
binding cysteine in the RNCIG motif, and so we
undertook molecular modelling to examine its struc-
ture. The model was based on the crystal structure of
the fatty-acid binding P450 from Bacillus megaterium
CYP102 (PDB code 1FAG) [27], with which it shares
a 21% amino acid identity. The model suggests that
CYP4x1 has an active site cavity that is rather differ-
ent in shape from that of CYP102 (Fig. 2).
The expression of CYP4x1 was investigated by nor-
thern blotting using an EcoRI cDNA fragment from
bases 91–1389 of the cDNA which was radiolabelled
by random priming. As shown in Fig. 3A, there was
expression of CYP4x1 RNA in brain, heart and kid-
ney, and lesser expression in skeletal muscle and liver,
and no detectable expression in other tissues. However,
probing of a blot of heart tissues showed that there
was minimal expression in heart, but high level expres-
sion in aorta (Fig. 3B). Sixty-five CYP4x1 ESTs were
detected in a database search (07 ⁄ 05), and of these, 18
were from brain, three were from aorta and 21 were
from tumours or cell lines, confirming the importance
of brain and aorta as sites of expression.
Cloning of mouse Cyp4x1
To be able to work with an experimentally tractable
species, the mouse Cyp4x1 cDNA was cloned from
brain RNA of 129S4 ⁄ Jae mice, based on the mouse
genomic sequence (AJ297131). A full-length cDNA
was cloned and sequenced (EMBL Accession number

in Fig. 4C, heart, lung, kidney and spleen showed very
low levels of expression, whereas there was expression
in aorta (Fig. 4D). The highest levels of expression
were shown in brain (Fig. 4E) from 129S4 ⁄ Jae mice,
and the levels of expression were comparable in brain
RNA from PPARa– ⁄ – mice. Treatment of mice
with the classical inducers 2,3,7,8-tetrachlorodibenzo-
p-dioxin, phenobarbital, dexamethasone or ciprofi-
brate, had no effect on the expression of Cyp4x1 in
brain (data not shown).
M. Al-Anizy et al. Cyp4x1 in mouse brain
FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS 937
A
B
Fig. 1. Alignment of CYP4 family sequences. (A) Mammalian CYP4 protein sequences were optimally aligned, then displayed using GENEDOC,
with black squares at 90%, dark grey squares at 75% and light grey square at 60% amino acid identity. Sequences are identified by gene
name, with the exception of rabbit CYP4B1 (cyp4b1), rat CYP4B1 (cyp4b1) and human CYP4B1 (cyp4b1). The conserved region is under-
lined. (B) Alignment of deduced amino acid sequence of rat, human and mouse CYP4x1 proteins.
Cyp4x1 in mouse brain M. Al-Anizy et al.
938 FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS
Expression of Cyp4x1 protein
To analyse the expression of Cyp4x1 protein, a full-
length clone of mouse Cyp4x1 was inserted into the
pRSETa expression vector (Invitrogen, Paisley, UK),
and insoluble recombinant protein was affinity purified
from bacteria (Fig. 5A). Polyclonal antisera raised
against the human CYP4x1 [25] bound to the mouse
protein with low affinity, and could not be used for
studies in mice (data not shown). The mouse Cyp4x1
fusion protein was used to raise antisera in rabbit,

(Fig. 5C). This shows that the antisera specifically
detect the murine Cyp4x1 protein. Serial dilution of the
purified recombinant antigen and brain microsomes
showed that there is  10 ng Cyp4x1 proteinÆmg
)1
brain microsomal protein, or 200 pmol Cyp4x1Æmg
)1
microsomal protein (Fig. 5D). This suggests that
Cyp4x1 is a major brain cytochrome P450 form [23].
Cyp4x1 immunohistochemistry in brain
The primary antiserum was used to locate Cyp4x1
protein by immunocytochemistry of paraformaldehyde
fixed wax embedded sections of 129S4 ⁄ Jae mouse
brain. Specific staining for Cyp4x1 protein was found
in the Purkinje cells of the cerebellum, pyramidal neu-
rons in the dentate gyrus of the hippocampus; cortical
forebrain neurons and those of brain stem nuclei; addi-
tionally choroid epithelial cells of the chroroid plexus
were also stained; (Fig. 6C–H). Control sections of
brain regions incubated without primary antiserum
failed to show specific staining (Fig. 6A) as did sec-
tions incubated with preadsorbed primary antiserum
(Fig. 6B). The brown 3,3¢-diaminobenzidine (DAB)
staining was granular and confined to the cytoplasm
of the cells. Based on their size and shape the cells
labelled in all brain regions appeared to be neurons.
Blood vessels were also stained showing that the vascu-
lar endothelial cells contained cyp4x1 protein.
Discussion
The conserved sequence identified by Heng [4] is speci-

gel. The tracks are numbered: 1, right ventricle; 2, left ventricle; 3,
right atrium; 4, left atrium; 5, apex of heart; 6, aorta; 7, heart; 8,
foetal heart. Both blots were hybridized with a human CYP4x1
probe, and autoradiographed. The position of the 2.2-kb human
CYP4x1 transcript is indicated by a horizontal line.
Cyp4x1 in mouse brain M. Al-Anizy et al.
940 FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS
AB
E
DC
Fig. 4. RNAase protection assay for Cyp4x1 RNA in 129S4 ⁄ Jae mice. (A) Cyp4x1 probe was hybridized to 30 lg of yeast tRNA in the pres-
ence (+) or absence (–) of added RNAase, or to 30 lg of Brain (B) or liver (L) RNA for RNAase protection assay. M indicates the 124-base
pair marker transcript (M), the full-length probe is indicated by a line, and the protected fragment is indicated by an arrow. The RNAase pro-
tection assay was performed as described in Experimental procedures, and the dried gel was exposed to film overnight. The vertical line
indicates that several tracks have been removed from the autoradiograph. (B) Animals were treated with vehicle, TCDD, phenobarbital (PB),
ciprofibrate (Cipro) or dexamethasone (Dex), as described in Experimental procedures, RNA isolated from liver, and 30 lg of RNA subjected
to protection assay. The film exposure was for 5 days. (C) Heart, kidney, lung and spleen RNA from each of three animals was analysed for
Cyp4x1 RNA, and a 5-day exposure of the autoradiograph is shown; – and + represent yeast tRNA without and with RNAase treatment. (D)
Thirty milligrams of RNA from pooled aorta from six mice treated with vehicle (Veh) or ciprofibrate (Cip) was analysed by RNAase protection,
and the results of an overnight autoradiograph are shown. (E) RNA was isolated from brain of untreated wild-type (+ ⁄ +) or PPARa nullizy-
gous (– ⁄ –) 129S4 ⁄ Jae mice, and 30 lg of RNA subjected to protection assay. The film exposure was overnight.
M. Al-Anizy et al. Cyp4x1 in mouse brain
FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS 941
21% amino acid identity, and good correlation
between observed secondary structural elements in
CYP102 and predicted elements in CYP4x1 (Fig. 2A).
Moreover, this model reveals an extended substrate
binding pocket (Fig. 2B) that suggests that CYP4x1 is
designed to bind substrates distinct in structure from
the medium-chain fatty acids bound by CYP102. The

ing a physiological effect, and so the lack of induction
of Cyp4x1 must reflect a lack of inducibility of this
gene in mouse liver (Fig. 4B). In agreement, there was
no evidence for induction of Cyp4x1 in aorta or brain
(Fig. 4D, and data not shown). This is in contrast to
the work of Savas, who reported that CYP4x1 was
inducible by peroxisome proliferators in human hepa-
toma cells transfected with PPARa [25]; however, it is
widely accepted that human liver cells do not induce
CYP4 genes in response to peroxisome proliferators
[32–34], and so the relevance of their observation is
open to question.
Although the distribution of CYP4x1 RNAs has
been examined, there is no data as to whether the
corresponding protein is translated in vivo. An antibody
specific for the mouse Cyp4x1 protein was developed,
as an antibody raised against human CYP4x1 showed
poor cross-reactivity against recombinant expressed
mouse Cyp4x1. The antibody detected 1 ng of recom-
binant antigen (data not shown), and detected a protein
of  55 kDa in brain microsomes, but failed to detect
M B U P S 4x
43
66
97
116
A
C
B
M 5 10 5 10 5 10 5 10 µg

Cyp4x1 in mouse brain M. Al-Anizy et al.
942 FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS
A
B
C
D
E
F
G
H
Fig. 6. Immunohistochemistry of Cyp4x1 in mouse brain. Male 129S4 ⁄ Jae mice were killed, and brain taken and fixed, and sections ana-
lysed by immunohistochemistry with an antibody against Cyp4x1, as described in Experimental procedures; staining shows as a brown
deposit. (A) No primary antibody. (B) Primary antibody was preadsorbed with Cyp4x1 antigen. (C) Dentate cells of the hippocampus. (D)
Outer layer of the hippocampus. (E) Choroid plexus. (F) Purkinje cells. (G) Brain stem. (H) Cerebral cortex. A scale bar is shown.
M. Al-Anizy et al. Cyp4x1 in mouse brain
FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS 943
this protein in liver microsomes (Fig. 5B), consistent
with the expected size of the P450, and the pattern of
distribution of the corresponding mRNA. The 55-kDa
protein was not detected by preimmune serum suggest-
ing that this is a specific reaction. To confirm this, the
antiserum was preadsorbed with the recombinant
Cyp4x1 antigen, and this ablated binding to the 55-kDa
protein, confirming that this protein is Cyp4x1. Serial
dilution of the Cyp4x1 protein showed that there is
 200 pmols of Cyp4x1Æmg
)1
microsomal protein; this
must be contrasted with the total P450 content of rat
brain, at  100 pmolsÆmg

129S4 ⁄ Jae PPARa+ ⁄ + and 129S4 ⁄ Jae PPARa– ⁄ – mice
were obtained from J.M. Peters (Department of Veterinary
Science, Pennsylvania State University, PY, USA) [29,35],
and maintained as a colony in house; animals were regularly
genotyped for PPARa status [5]. Vehicle treated mice (n ¼
3) received a single dose by gavage of 10 mLÆkg
)1
of corn
oil, containing 2.5% (v ⁄ v) p-dioxane, and mice treated with
2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) received the
same vehicle, but containing 50 lg TCDDÆkg
)1
bodyweight.
Phenobarbital was administered in saline as an i.p. injection
of 80 mgÆkg
)1
bodyweight daily for 3 days; ciprofibrate and
dexamethasone were dissolved in corn oil, dosed at
50 mgÆkg
)1
bodyweight by gavage daily for 3 days; all ani-
mals were killed on day 4. Tissues were frozen in liquid N
2
,
prior to storage at )80° C for the isolation of RNA by the
Triazol method (Invitrogen), or used directly for the prepar-
ation of microsomes. New Zealand White rabbits were used
for immunization with Freund’s incomplete adjuvant, and
subsequent monthly boosters.
All experiments on animals were conducted under the

RNAase protection
The RNase protection assay used the Riboquant
Ò
kit, from
PharMingen Ltd (Oxford, UK), according to the manufac-
turer’s instructions. Briefly, template DNA was linearized
with NcoI, then transcribed using [a-
32
P]CTP. The probe
was treated with DNAse I, then proteinase K, phenol ⁄ chlo-
roform extraction, and ammonium acetate ⁄ ethanol precipi-
tation. Thirty micrograms of each RNA sample was
precipitated, resuspended with 8 lL hybridization buffer,
1 lL of synthesized probe added, then incubated overnight
Cyp4x1 in mouse brain M. Al-Anizy et al.
944 FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS
at 56 °C. The samples were treated for 45 min at 42 °C
with 100 lL RNase cocktail, except the positive control
(–ve tRNA) which was treated with 100 lL buffer. Eighteen
microlitres of proteinase k cocktail were added and the
reactions were incubated for 15 min at 42 °C. Samples were
extracted with phenol ⁄ chloroform, then ethanol precipita-
ted and electrophoresed on a denaturing 6% acrylamide
gel, followed by autoradiography.
Western blotting
Western blotting was performed essentially as described
[36]. For preabsorption with antigen, the antisera was dilu-
ted 1 : 500 in Tris-buffered saline pH 7.4, containing 0.1%
Tween and 10% Marvel, then 50 lg purified Cyp4x1 anti-
gen were added, followed by incubation for 1 h at 4 °C.

dase (HRP) secondary antibody system from DAKO
LSAB2 kit (DakoCytomation, Ely, UK) using the bio-
tin ⁄ streptavidin system with DAB (DakoCytomation) as
chromogen. The sections were preincubated for 10 min in
hydrogen peroxide to quench endogenous peroxidases and
blocking serum then incubated with primary antibody for
2 h followed by HRP-labelled secondary antibody (LSAB 2
kit) and finally incubated with DAB solution for 10 min.
The sections were mounted in Vectashield prior to examina-
tion in a computer linked Leitz photo-microscope (Leica
Microsystems, Milton Keynes, UK). The negative control
sections were incubated without primary antibody or with
primary antiserum preadsorbed with purified P450 Cyp4x1
protein and treated as for test sections.
Molecular modelling
The alignment of the CYP4x1 and CYP102 amino acid
sequences was performed using clustalw [37] and refined
manually in the light of the comparison of secondary struc-
ture data ) from the crystal structure in the case of
CYP102 – and predicted using jpred [38] in the case of
CYP4x1. Coordinates for the main chain atoms of aligned
CYP4x1 residues were taken directly from their CYP102
counterparts, and initial side chain coordinates were
assigned using the rules of Summers and Karplus [39].
Main chain coordinates for residues in loops were obtained
using the search method implemented in sybyl (Tripos
Inc., St Louis, MO, USA). All side chain conformations
were then re-predicted using rascle, in-house software
based on the method of local environment similarity [40].
Finally, the model was refined by molecular mechanics

FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS 945
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procedures.
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from
M. Al-Anizy et al. Cyp4x1 in mouse brain
FEBS Journal 273 (2006) 936–947 ª 2006 The Authors Journal compilation ª 2006 FEBS 947