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] proteins are known to have fatty acid hydroxylase activity, and there is extensive speculation that the formation of hydroxylated fatty acids by cytochrome P450 leads to the production of physiologically active metabolites that regulate physiological function [11–16]. Cytochrome P450 metabolism of fatty acids may also be of fundamental importance in brain [17–19], and it is known that neurotransmitters and fatty acids can be actively metabolized by cytochrome P450 in brain [18,20,21]. Although the specific content of cytochrome P450 in brain is relatively low compared with liver [17,22,23], this level of cytochrome P450 can be induced by various agents [24]. A peculiar feature of brain P450 is that it is difficult to account for the total P450 con- tent with previously characterized P450 proteins [17]. We describe the cloning of human and mouse cDNAs for the CYP4x1 P450, a molecular model of the protein, and tissue-specific localization of the RNA in mouse and human. The Cyp4x1 protein was locali- zed by immunohistochemistry and shown to be a major P450 protein in mouse brain. Keywords Cytochrome P450; peroxisome proliferators; brain; aorta Correspondence D.R. Bell, School of Biology, University of Nottingham, University Park, Nottingham. NG7 2RD, UK Fax: +44 115 9513251 Tel: +44 115 9513210 E-mail: david.bell@nottingham.ac.uk (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 We previously noted that members of the murine Cyp4a subfamily showed high conservation of the protein sequence in exon 4, amino acids 125–167 of Cyp4a10 [4]. A more comprehensive alignment of CYP4 family proteins was undertaken (Fig. 1A), which confirmed the high conservation of this region of the protein sequence. CYP4A1 has been modelled based on CYP102 (C.A. Laughton, unpublished data), and this model suggests that this region is a part of the C-helix, which may be involved in making conserved contacts with conserved residues in the I-helix. To test whether this sequence was conserved, we used a BLAST search of Uniprot (http://www.ebi.uniprot.org, June 2005) with residues 124–158 of rat CYP4A1, which detected putative members of the CYP4 family. Interestingly, this search also detected unknown, putative human CYP4 transcripts, R53456, which was sequenced, leading to the identification of further EST clones, AA337301 and AA319338. The full-length cDNA for this gene was cloned by screening of a human aorta cDNA library, and RT ⁄ PCR, and yielded a full-length clone, which was then sequenced on both strands (EMBL Accession number AM040940). This cDNA encodes an ORF of 509 amino acids, a predic- ted molecular mass of 58 874 Da, a pI of 8.5, and has been designated CYP4x1 on the basis of 70% amino acid identity to the rat CYP4x1 (Fig. 1B). While this work was in preparation, this sequence has been cloned [25] and described in bioinformatics analysis [26] by others. 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 AJ786769), encoding a putative protein of 507 amino acids, a molecular weight of 58 556 Da, a pI of 7.19, with 94 and 71% identity with the rat and human CYP4x1 proteins, respectively (Fig. 1B). A genomic fragment of the CYP4x1 was cloned, containing 247 bp of intron and 177 bp of exon, and was used for RNAase protection assays. The specificity was con- firmed by the finding that yeast tRNA failed to protect the probe sequence (Fig. 4A), but there was high-level specific protection of 177 bp of the probe by mouse brain RNA, whereas liver RNA showed very low-level protection of the 177-bp fragment. The protection of a fragment of 177 bp demonstrates that the protection of the probe is specific for the size of the exon frag- ment, and is therefore specifically detecting the expres- sion of Cyp4x1 RNA. The inability of yeast tRNA to protect the Cyp4x1 probe shows that the RNAase protection is highly specific, consistent with previous reports (e.g. [3,28]). Numerous members of the CYP4 family are known to show liver-specific expression or induction (e.g. in mouse [3,4]), so we determined if the Cyp4x1 RNA was inducible by treatment of mice with the classical inducers 2,3,7,8-tetrachlorodibenzo- p-dioxin, phenobarbital, dexamethasone or ciprofi- brate. As shown in Fig. 4B, there was a low level of expression of Cyp4x1 in control mice, but no hepatic induction of Cyp4x1 RNA after treatment with these inducers, nor was the level of Cyp4x1 RNA perturbed in liver RNA from PPARa– ⁄ – mice [29]. Extrahepatic expression of Cyp4x1 was also investigated. As shown 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, which were able to detect  1 ng of the antigen (data not shown). As shown in Fig. 5B, antisera detected a principal protein band of  55 kDa in brain micro- A B Fig. 2. Modelling of human CYP4x1. (A) Sequence alignment of CYP4x1 with the bacterial crystal structure, CYP102 (PDB code 1FAG, chain B). Highlighted regions in green represent a-helix and red regions rep- resent b-sheet (predicted in the case of CYP4x1 and known for the crystal structure in the case of CYP102). Numbers at the side of the alignment represent the residue number. (B) Molecular model of the predic- ted active site cavity for CYP4x1, produced using GRASP [46] and colour-coded by elec- trostatic potential (red: negative, blue: posit- ive). For clarity, only the position of the haem 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 939 somes, but not in liver microsomes, although several other minor bands were detected. This 55-kDa band was not detected by preimmune serum from the same rabbits (data not shown). To determine if this represen- ted specific binding, the antisera were preincubated with purified recombinant Cyp4x1 protein, resulting in the ablation of detection of the 55-kDa protein in brain (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- fic for the CYP4 family, detecting numerous members of this family. The sequence at amino acids 124–158 has been modelled to contain the C-helix [30,31], with conserved residues contacting the I-helix (C.A. Laugh- ton, D.R. Bell, unpublished data). The high conser- vation of this region is evidenced by the specific detection of cytochromes P450, and in particular, members of the CYP4 family. It was therefore of great interest when two human ESTs were detected, corres- ponding to a novel cytochrome P450. Cloning of the full-length cDNA revealed that the corresponding pro- tein was a member of a new subfamily, CYP4x1, and showed high sequence identity to the previously repor- ted rat CYP4x1 sequence [26], and perfect identity to the protein sequence reported by Savas [25]. The predicted protein sequence shows the expected features of a cytochrome P450, including the canonical haem-binding cysteine motif, and conservation of other conserved features [30,31]. Molecular modelling confirmed that the sequence has a primary structure consistent with a functional, P450 structural fold. The CYP4x1 and CYP102 sequences can be aligned with A B Fig. 3. Northern blotting of human CYP4x1 RNA. (A) Approximately 1 lg of each poly A+ RNA was run on a denaturing 1% agarose gel. The tracks are: 1, brain; 2, heart; 3, skeletal muscle; 4, colon; 5, thymus; 6, spleen; 7, kidney; 8, liver; 9, small intestine; 10, pla- centa; 11, lung; 12, peripheral blood leucocytes. (B) Approximately 2 lg of each poly A+ RNA was run on a denaturing 1% agarose 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 pocket is approximately L-shaped, with the haem located at the angle. This suggests that the substrates may either be shorter chain fatty acids that might bind in one of two possible orientations, or alternatively longer-chain fatty acids, but that the oxidation of these is not directed, as is usually the case in the CYP4s, to the terminus of their carbon chain, but rather to some position mid-way along the chain. Northern blot analysis showed high level expression of the CYP4x1 RNA in brain and in aorta, and this was confirmed by analysis of the EST database; this showed significant numbers of CYP4x1 ESTs in brain and aorta. An unexpected finding is that there was a high number of CYP4x1 ESTs from tumour tissue, suggesting that CYP4x1 may have some role in tumo- rigenesis. Further analysis of the function of CYP4x1 was pre- cluded by the difficulty in obtaining human tissue sam- ples, and the mouse Cyp4x1 was therefore cloned from brain RNA of 129S4 ⁄ Jae mice. This sequence had 94% identity to the rat sequence, and was confirmed as the mouse Cyp4x1 on the basis of sequence identity. RNAase protection assay showed that the RNA was expressed at high level in brain and in aorta, but that there were much lower levels in other tissues. Our results showed no induction of Cyp4x1 RNA in liver after treatment with the potent peroxisome prolifera- tors, ciprofibrate, at a dose which caused liver enlarge- ment (data not shown). Since this dose of peroxisome proliferators causes liver enlargement, it is clearly hav- 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 Brain Liver Liver Brain α-mCyp4x1 pre-adsorbed 30 10 ng Cyp4x1 3 1 0.5 0.1 µg microsomes Fig. 5. Western blotting for mouse Cyp4x1. (A) Expression and purification of the Cyp4x1 antigen. Total protein from BL21(DE3) cells (B) or uninduced cells containing the pRSET-mCyp4x1 (U), the 10 000-g pellet from cells induced for 3 h with 1 mm isopropyl thio-b- D-galactoside (P), and the proteins solubilized from the pellet by extraction with 8 M urea (S) were prepared as described in Experimental procedures, and 20 lg electrophoresed on an SDS ⁄ PAGE gel. Cyp4x antigen purified on a nickel-affinity column is shown in track 4x. The molecular mass of markers (M) is shown in kDa. (B) Western blotting for mCyp4x1. Five and 10 lg brain and liver microsomes were eletrophoresed on SDS ⁄ PAGE, blotted and developed with antimCyp4x1 antibody, or antibody that had been preadsorbed with 80 lg of the mCyp4x1 antigen. The marker (M) is the histidine-tagged mCyp4x1 protein, and its mobility is indica- ted by a line; the mobility of the Cyp4x1 specific band is indicated by an arrow. (C) Quantification of Cyp4x1 in brain microsomes. Thirty and 10 ng of purified recombinant Cyp4x1 was run on the same gel as the indicated amount of brain microsomes, western blotted and chemiluminescence recorded on film. The mobility of histidine-tagged Cyp4x1 antigen is indicated by a line; the mobility of the Cyp4x1-specific band is indicated by an arrow. 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 )1 of microsomal protein. It has previously been shown that P450 may be present as both the holoenzyme and the apoenzyme; however, we have not determined how much of the mouse Cyp4x1 protein is present as holoenzyme in mouse brain. Further characterization of the Cyp4x1 in mouse brain showed that the P450 was expressed in the cyto- plasm of neurons in the cerebellum, and in the vascu- lar endothelium. This staining could be shown to be specific for Cyp4x1, as controls lacking primary anti- body, or which had been preadsorbed with excess Cyp4x1 antigen, showed no specific staining of these cells; additionally, there was specificity in the staining of neuronal cells in the cerebellum, as Purkinje cells were not stained for Cyp4x1. These results extend and confirm a previous report of localization of CYP4x1 RNA in rat brain [26]. The high level expression of Cyp4x1 in brain and aorta begs the question of the functionality of this pro- tein, and its physiological role. We aim to conduct future experiments to determine the enzymatic activity of this P450, and how this relates to physiological function. Experimental procedures Animals and tissue Human cardiovascular system and 12-lane multitissue nor- thern blots, human aorta cDNA library and RACE ready aorta cDNA were obtained from Clontech (Oxford, UK). Human IMAGE clones ID 139602 and 119884 were obtained from ATCC (LGC Promochem, Teddington, UK). 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 authority of a project licence granted by the Home Office to D.R. Bell, in accordance with UK law. cDNA cloning EST cDNA clones for R53456 and AA337301 were obtained from HGMP (Hinxton, Cambridgeshire, UK), and sequenced on both strands. The latter clone was diges- ted with EcoRI and the 1.2-kb fragment was radiolabelled with random primers for screening an aorta cDNA library. Full-length clones were confirmed by sequencing. For pro- duction of human CYP4x1 antigen, the cDNA was digested with BamHI and BstBI, and the fragment cloned into pRS- ETc (Invitrogen) for expression of amino acids 327–509 of CYP4x1 as an N-terminally histidine-tagged fusion protein in bacteria. A probe for RNAase protection for mouse Cyp4x1 was obtained by PCR of brain RNA with primers 4x1-12-pF, 5¢-CATGGACATAAGTCCTTTTCCCTTCCTCCT-3¢, and 4x1-12-pR, 5¢-AAACATAAATTTCGCCATTTCTCCTAG TAT-3¢. The full-length mouse cDNA was obtained with primers 4x1-f-pF, 5¢-ATGGAGGCCTCCTGGCTGGAG ACTCGTTGG-3¢ and 4x1-f-pR, 5¢-AAACATAAATTT CGCCATTTCTCCTAGTAT-3¢. Total RNA from mouse brain was used, and ProSTAR UltraHF RT ⁄ PCR Kit was used to generate the first strand cDNA. PCR products were cloned into pGEM-T (Promega), and sequenced on both strands. The full-length clone was cloned into pRSETa for bacterial expression of antigen. Antigen was expressed by recombinant expression in BL21(DE3) bacteria (Novagen, Merck Biosciences, Nottingham, UK), and affinity purifica- tion of pelleted insoluble protein in 8 m urea. 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. The same procedure was followed without the addition of antigen for the nonpreadsorbed antisera. These antisera were then used as primary antisera in the western blotting process. Microsome preparation Organs were homogenized in a motor-driven Teflon pestle and glass tube in ice-cold 0.15 m NaCl, 10 mm KH 2 PO 4 , pH 7.4. After centrifugation for 10 min at 10 000 g, sam- ples were centrifuged at 400 000 g for 30 min, and the pel- let resuspended in 20% glycerol (w ⁄ v), 0.1 m KH 2 PO 4 , pH 7.4. Samples were normalized by Bradford assay, relat- ive to BSA. Immunohistochemistry The brains were fixed for immunocytochemistry by trans- cardiac perfusion of anesthetized male 129S4 ⁄ Jae mice (lethal dose of Nembutal i.p.) firstly by prewash saline solu- tion at 37 °C followed by 4% freshly prepared paraformal- dehyde in 0.1 m phosphate buffer pH 7.4 at 5 °C. The brains were dissected out and embedded in paraffin wax. Sections of 10 lm thickness were cut and de-waxed prior to incubation with primary antibody. The antigen was visualized using the horseradish peroxi- 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 using amber [41]. The quality of the model was checked using procheck [42] and prosaii [43]. Two further models were also built, to check consistency in the predictions. Model 2 used an alternative approach, scwrl [44], for the side chain prediction; model 3 used the modeller [45] package throughout, from the point of the sequence align- ment. The three final models all showed the same general features with regard to the shape and size of the binding cavity, so only results for model 1 are shown here. Acknowledgements Mohammed Al-anazy was supported by a scholarship from the Saudi government, and this work was supported by a grant from the Wellcome trust (054778 ⁄ Z ⁄ 98). We wish to thank Declan Brady for invaluable technical support and assistance. References 1 Nelson DR et al. (1996) P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42. 2 Coon MJ (2005) Cytochrome P450: Nature’s most ver- satile biological catalyst. Annual Review of. Pharmacol Toxicol 45, 1–25. 3 Bell DR et al. 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Cyp4x1 protein mg )1 microsomal protein, showing that Cyp4x1 is a major brain P450. Immunohistochemical localization of the Cyp4x1 protein in brain showed. localization of the RNA in mouse and human. The Cyp4x1 protein was locali- zed by immunohistochemistry and shown to be a major P450 protein in mouse brain. Keywords Cytochrome