Ilka U Heinemann

Nina Diekmann

Ava Masoumi

Michael Koch

Albrecht Messerschmidt

Martina Jahn

Dieter Jahn
email: d.jahn@tu-bs.de

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate binding site

Keywords: Heme biosynthesis, Protoporphyrin, Mutant variants, Variegate Porphyria, Enzyme Kinetics, Stopped-Flow Fluorescence PPO, protoporphyrinogen IX oxidase, PPO2, mitochondrial protoporphyrinogen IX oxidase, protogen, protoporphyrinogen IX, proto, protoporphyrin IX, VP, Variegate Porphyria, PPO1, plastidic

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in heme and chlorophyll biosynthesis. The apparent K m value for wildtype tobacco PPO2 (mitochondrial PPO) was 1.17 µM, with a v max of 4.27 µM min -1 mg -1 and a catalytic activity k cat of 6.0 s -1 . Amino acid residues that appear important for substrate binding in a crystal structure based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe 392 in substrate ring A stacking. Mutations of Leu 356 , Leu 372 and Arg 98 increased k cat values up to 100-fold indicating that the native residues are not essential for establishing an orientation of the substrate conducive to catalysis. Increased K m values of these PPO2 variants from 2 -100-fold suggest that these residues are involved but not essential to substrate binding via ring B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore, tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.

INTRODUCTION

PPO (protoporphyrinogen IX oxidase, EC 1.3.3.4) catalyses the oxygen-dependent aromatisation of protogen (protoporphyrinogen IX) to proto (protoporphyrin IX) [START_REF] Brenner | The enzymatic defect in variegate porphyria. Studies with human cultured skin fibroblasts[END_REF] (Fig. 1). It is the last common step of tetrapyrrole biosynthesis for the formation of hemes and chlorophylls. Initially, the enzyme was characterised from yeast [START_REF] Poulson | The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae[END_REF] and mammalian liver [START_REF] Poulson | The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria[END_REF][START_REF] Dailey | Purification and characterization of murine protoporphyrinogen oxidase[END_REF]. It catalyses the six-electron oxidation of protogen using a flavin cofactor and molecular oxygen as terminal electron acceptor. PPO is described as soluble monomer in Bacillus subtilis [START_REF] Hansson | Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes[END_REF], as membrane-associated monomer in bovine [START_REF] Siepker | Purification of bovine protoporphyrinogen oxidase: immunological cross-reactivity and structural relationship to ferrochelatase[END_REF] or as homodimer in Myxococcus xanthus and humans [START_REF] Dailey | Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme[END_REF][START_REF] Dailey | Expression, purification, and characteristics of mammalian protoporphyrinogen oxidase[END_REF][START_REF] Maneli | Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases[END_REF]. In eukaryotes PPO is associated with the outer surface of the inner mitochondrial membrane [10], while the plant enzyme is additionally located in chloroplasts [START_REF] Jacobs | Protoporphyrinogen oxidation, an enzymatic step in heme and chlorophyll synthesis: partial characterization of the reaction in plant organelles and comparison with mammalian and bacterial systems[END_REF][START_REF] Dailey | Terminal steps of haem biosynthesis[END_REF].

In humans, partial PPO deficiency causes a disease known as VP (variegate porphyria, OMIN TM accession number 176200) that is inherited as an autosomal dominant trait displaying incomplete penetrance [13]. The biochemical abnormalities found in VP patients include overproduction and increased excretion of porphyrins and porphyrin precursors. VP manifests clinically with photosensitivity and acute attacks, which includes various neuropsychiatric symptoms [14]. In South Africa VP is common due to a founder effect, where mainly one amino acid exchange from Arg 59 to tryptophan causes the disease [15][16][17][START_REF] Roberts | Molecular characterization of homozygous variegate porphyria[END_REF]. However, there are various additional mutations known to cause VP [START_REF] Fraunberg | Clinical and biochemical characteristics and genotype-phenotype correlation in Finnish variegate porphyria patients[END_REF].

In plants two isoforms of PPO, PPO1 (plastidic PPO) and PPO2 (mitochondrial PPO) have been found [START_REF] Jacobs | Protoporphyrinogen oxidation, an enzymatic step in heme and chlorophyll synthesis: partial characterization of the reaction in plant organelles and comparison with mammalian and bacterial systems[END_REF][START_REF] Lermontova | Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase[END_REF][START_REF] Beale | Enzymes of Chlorophyll biosynthesis[END_REF]. They share an amino acid sequence identity of less than 30%. Both enzymes operate as homodimers and use FAD as cofactor. Recently, the crystal structure of tobacco (Nicotiana tabacum) PPO2 with the bound inhibitor INH (4-bromo-3-(5'-carboxy-4'-chloro-2'-fluoro-pheny)-1methyl-5-trifluoromethyl-pyrazol) and non-covalently bound FAD has been solved [22]. This crystal structure is the only currently available structural information for eukaryotic PPO at the atomic level.

Copyright 2006 Biochemical Society

The structure of tobacco PPO2 revealed a loosely associated dimer, each monomer consisting of a membrane-, an FAD-and a substrate-binding domain, respectively. The inhibitor INH was found to be located in the active-site domain and consists of a phenyl-and pyrazol-ringsystem. Even though the FAD is not covalenty bound to the enzyme it was found in close proximity to the cocrystallized inhibitor in the active site cavity. A potential reaction mechanism was proposed based on the fact that the substrate has only limited space to rotate in the active site. The six electron oxidation of protogen to proto is supposedly catalysed via three steps. Three times the FAD cofactor is reduced by two electrons from the tetrapyrrole ring and reoxidised by molecular oxygen which in turn is reduced to H 2 O 2 . The reduction starts at the C 20 atom of the tetrapyrrole via enamine-imine-tautomerisation and H-rearrangements [START_REF] Phillips | Structural basis for tetrapyrrole coordination by uroporphyrinogen decarboxylase[END_REF].

Using a model calculated based on the solved crystal structure of tobacco PPO2, we have generated 14 variant proteins carrying amino acid residue exchanges in the corresponding positions of the active-site domain. The mutant variant proteins were kinetically characterised by steady state and stopped-flow fluorescence spectrometry. Furthermore, the amino acid residues responsible for the most common cause of VP in humans were replaced and analysed on the basis of the crystal structure of PPO2 from tobacco.

EXPERIMENTAL Materials:

Proto was purchased from Sigma-Aldrich (Hamburg, Germany). Tween 20 was obtained from Carl Roth GmbH (Karlsruhe, Germany). The remaining chemicals were purchased from Sigma-Aldrich unless specified otherwise. Mutagenesis experiments were performed using the QuikChange TM site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). Oligonucleotides were purchased from Biomers.net GmbH (Ulm, Germany). S•Tag Rapid Assay Kit was from Novagen (Darmstadt, Germany). Cells were disrupted using a sonotrode from Bandelin (Berlin, Germany). Protino ® Ni-IDA Resin was from Machery-Nagel (Düren, Germany).

Site-directed mutagenesis of tobacco PPO2 gene

The N. tabacum PPO2 gene expression vector pET32a-PPO2(M1-K224Q) was described before

PPO assays

The substrate protogen was produced as described before [START_REF] Labbe | Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase[END_REF][START_REF] Layer | Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes[END_REF]. PPO2 activity was monitored using a continuous assay via the detection of proto fluorescence. Stopped-flow kinetic experiments were performed under aerobic conditions using a Jasco 810 stopped-flow spectrometer and obtained data were analysed with Biokine Acquire Kinetics (Jasco, Überlingen, Germany). Averages of three to five individual transients were analysed. Protogen was dissolved in 50 mM Mes-buffer pH 6.0, 1 % (v/v) Triton X-100, 20 mM EDTA. Protogen solution and PPO2 preparations were mixed in a six to one ratio in a total volume of 300 µl and fluorescence resulting from proto formation was continuously recorded via an excitation at 409 nm and emission measurement above 450 nm.

Approximately 8000 measurement points per assay were recorded. Proto concentrations were calculated using an appropriate calibration curve. The percentage of substrate consumed was less than 5%.

Determination of kinetic constants

The Michaelis Menten constant (K m ), the maximal velocity v max and the catalytic constant k cat were determined from substrate velocity plots by measuring the constant velocity formation of proto from protogen over a substrate range from 1 -25 µM and a time course of up to 2 hours at 20°C. The initial reaction rates were linear and values were determined by the computerised Lineweaver Burke iterative curve fitting using SigmaPlot 8.0 Enzyme Kinetics v1.1. For the calculation of the catalytic efficiency k cat , the maximal velocity v max was divided by the corresponding enzyme concentration.

RESULTS AND DISCUSSION

Experimental determination of amino acid residues important for substrate binding

Based on the proposed model we functionally investigated the molecular basis of substrate coordination and its catalytic implications. We constructed an appropriate set of mutant PPO2 genes, isolated the respective mutant protein variants and determined their kinetic parameters via fluorescence stopped flow spectrometry in comparison to wildtype PPO2. We used the strategy of introducing conservative and non-conservative amino acid exchanges for residues of interest in order to evaluate their chemical contribution to substrate binding and catalysis. Obtained results are listed in table 1. The apparent K m value of purified wildtype PPO2 protein for its substrate was 1.17 µM with a k cat value of 6.0 s -1 . The determined kinetic parameters were within the range of values measured for PPOs from other organisms, e.g. K m = 1.0 µM, k cat = 0.0031 s -1 for B. subtilis [START_REF] Corrigall | Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis[END_REF], K m = 0.1 µM for Saccharomyces cerevisiae [START_REF] Camadro | Purification and properties of protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. Mitochondrial location and evidence for a precursor form of the protein[END_REF], K m = 2.8 µM for Aquifex aeolicus, [START_REF] Wang | Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus[END_REF] and K m = 0.85 µM and for Homo sapiens either a k cat = 0.175 s -1 [START_REF] Dailey | Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme[END_REF] or a k cat = 5.95 s -1 [START_REF] Maneli | Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases[END_REF], respectively. The FAD content of PPO2 was 0.63 moles FAD/subunit PPO2. All PPO2 mutant enzymes revealed a similar FAD content, the only exception being the double mutant N67W/S374D which is discussed in detail later (table 1).

The crystal structure revealed that ring A of the inhibitor INH is kept in position by aromatic stacking with residue Phe 392 , which during reaction stabilises the electron transfer over the ring. An amino acid exchange to Glu 392 in the variant F392E was tolerated within the active site cavity and resulted in inferior binding of the substrate protogen (K m = 11.2 µM). However, once the substrate protogen was bound, its conversion was catalysed sufficiently (k cat = 11 s -1 ). The tetrapyrrole ring is made positive by the hydride abstraction at the N5 atom of FAD. Thus the negative charge of the introduced glutamate might stabilise the ring and catalysis may proceed equally fast. In this case only the stabilisation of the substrate by aromaticity is lowered and so its binding is weaker. Additionally, the PPO2 variant F392H was found to be virtually inactive. Determination of reliable kinetic parameters was not possible. Histidine at a pH below its pK at 6.1 is positively charged. Here at a pH of 6.0, this holds true for most histidine molecules of PPO2. The positive charge of the histidine ring at this position lead to repulsion and obstructs substrate binding, consequently reactions kinetics collapsed (Fig. 2 A). For detailed kinetic parameters of the mutant proteins please consult table 1.

In the model for substrate binding deduced from the PPO2-INH crystal structure ring B of protogen is sandwiched between the conserved residues Leu 356 and Leu 372 . Both amino acid residues were exchanged to either valine or asparagine, respectively. In both cases the conservative amino acid exchange to valine enhanced catalysis up to 100-fold (k cat = 300 s -1 and k cat = 597 s -1 , respectively). However, in the case of an amino acid exchange of Leu 372 to valine binding of the substrate protogen was 100-fold reduced (K m = 103 µM). In contrast to that the PPO2 variants L356N and L372N showed an only slightly inferior binding capacity compared to the wildtype and revealed comparable catalytic PPO2 activities (see table 1). Valine possesses comparable hydrophobic features to leucine. However, the carbon backbone of the side chain is shorter than that of leucine. Most likely the appropriate location of the hydrophobic residue in relation to the coordinated substrate ring is essential, explaining the increasing K m values of L356V and especially L372V. Once the substrate is bound catalysis is found enhanced for both mutants. Most likely the smaller side chain of valine better accommodates the necessary complex arrangement of the substrate during catalysis. On the other hand asparagine obviously provides the necessary side-chain length and chemical character to supplement for leucine in both positions (Fig. 2A). The next investigated amino acid residue potentially involved in substrate binding was Arg 98 . It was deduced from the N. tabacum PPO2 crystal structure that Arg 98 provides the counter charge for the carboxylate group of the inhibitor which most likely mimics the propionate group of ring C of protogen. The PPO2 variants R98K, R98E and R98A were chosen to evaluate the contribution of this positively charged amino acid. While the conservative exchange R98K improved enzyme activity (k cat = 37 s -1 ), the non-conservative exchange R98E resulted in inferior substrate binding (K m = 12.5 µM). Most likely the introduced negative charge prohibited the ionic interaction necessary for the stabilisation of ring C. In agreement with this assumption the replacement of Arg 98 with the non polar amino acid alanine resulted in an enzyme variant (R98A) with an eight-fold increased K m value (Fig. 2 A). Interestingly, all three variants in position 98 showed an increased k cat value, especially variant R98A (k cat = 309 s -1 ), indicating improved turnover in the absence of ionic interaction between enzyme and substrate. Nevertheless the overall response of PPO2 towards the introduced amino acid exchanges in position 98 is nicely reflected by the k cat and K m values.

In summary, by measuring enzyme activity of mutant PPO2s we were able to verify that the well conserved residues Arg 98 , Phe 392 , Leu 356 and Leu 372 are functionally involved in substrate coordination within the active site.

Variegate Porphyria

The dominantly inherited genetic disorder VP in humans results from impaired PPO activity as a consequence of an amino acid residue exchange. In more than 94% of the VP cases in South Africa, Arg 59 is mutated to tryptophan [16]. The corresponding residue to human Arg 59 in the tobacco PPO2 is Asn 67 . It is positioned on a longer loop between a β-strand and an α-helix exactly between the FADbinding and substrate-binding sites (Fig. 2 B). The residue is conserved in all known mitochondrial and plastidic plant PPO amino acid sequences. We started to mimic the situation of the human enzyme on the basis of the related crystal structure of PPO2 from tobacco by mutating Asn 67 to arginine. The obtained mutant N67R was found to be reasonably active. Even though the mutation resulted in inferior substrate binding, the catalytic efficiency was acceptable. This observation is in agreement with the findings of Maneli et al. [START_REF] Maneli | Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases[END_REF] Copyright 2006 Biochemical Society abolished enzyme activity. The bulky nature of an aromatic tryptophan in position N67 results in impaired enzyme activity. Thus, the enzymatic defect responsible for most VP cases in humans can be transferred onto tobacco PPO2. The spatial structure of PPO2 from tobacco can be used as a model for human PPO. However, in the tobacco PPO2 crystal structure the residue opposite to Asn 67 is Ser 374 (Fig. 2 B). The corresponding residue in human PPO is Asp 349 conserved in all known animal PPOs.

Asp 349 is likely to form a salt bridge with Arg 59 , which would sterically fit in this area of the protein [22]. In order to further adapt tobacco PPO2 towards the human counterpart, we first mutated Ser 374 to aspartate. The S374D single mutant showed reduced substrate binding capacity with a significantly increased k cat value (k cat = 208 s -1 ). Subsequently, we mutated Asn 67 of S374D to arginine to reconstruct the assumed salt bridge Asp 374 /Arg 67 in the human enzyme for our tobacco VP model. The resulting N67R/S374D double mutant showed an almost wildtype level K m value (K m =1.4). We were able to obtain a PPO2 variant with wildtype like substrate recognition and even improved catalytic properties (k cat = 111 s -1 ) by a reconstruction of the salt bridge in the tobacco enzyme. These results argue for the existence of a salt bridge linking Arg 59 and Asp 349 in the human enzyme as well. The double mutation N67W/S374D, mimicking VP in the human enzyme significantly reduced enzyme activity. The K m value was increased by a factor of 20 while the k cat /K m value decreased 10 fold.

Obviously the also reduced FAD content of 0.38 moles FAD/subunit for PPO2 variant N67W/S473D representing approximately 60 % of the wildtype enzyme content does not account for the observed decrease in enzyme activity. Similar observations were made for the human R59W mutant enzyme before [START_REF] Maneli | Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases[END_REF]. Most importantly, in contrast to most other generated mutant enzymes this PPO2 variant showed an increased K m with a parallel decreased k cat compared to the double mutant N67R/S374D. Both amino acids neighbouring Asp 349 (Ser 374 in PPO2) in the human enzyme have previously been characterised. A mutation of tryptophan in position 348 of the human enzyme to cysteine resulted in a wildtype-like K m value and a tenfold reduced k cat [START_REF] Maneli | Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases[END_REF]. A mutation of Ser 350 to proline abolished enzyme activity [START_REF] Roberts | Molecular characterization of homozygous variegate porphyria[END_REF]. These observations underscore that the structural integrity of this region is important for enzyme activity. Protogen is oxidised to proto by molecular oxygen with the generation of hydrogen peroxide.

Hydrogen atoms eliminated during the reaction are written in bold letters.s Residue Phe 392 is shown to coordinate ring A of the protogen. Leu 356 and Leu 372 are stacking protogen ring B from the "back" and the "front", Arg 98 is supposed to coordinate the propionate carboxylate group of the protogen ring C, respectively. (B) Variegate Porphyria mutants of tobacco PPO2.

Residues Asn 67 and Ser 374 playing an important role in human VP are depicted.

[

  22]. The plasmid was used as template for site-directed mutagenesis. Primers to generate N67R, N67W, R98A, R98E, R98K, L356N, L356V, L372N, L372V, S374D, F392E, and F392H, respectively, were as follows: N67R 5´-GGGATGAAGGGGCACGTACTATGACTG-3´ and 5´-CAGTCATAGTACTTGCCC-CTTCATCCC-3´; N67W 5´-GGGATGAAGGGGCATGGACTATGACTG-3´ and 5´-CAGTCATA-GTCCATGCCCCTTCATCCC-3´; R98A 5´-CCACTTTCACAAAACAAGGCCTACATTGCCAG-Biochemical Journal Immediate Publication. Published on 30 Nov 2006 as manuscript BJ20061321 Copyright 2006 Biochemical Society

  Active site architecture of N. tabacum PPO2. The crystal structure of tobacco PPO2 revealed a close insight into the three-dimensional structure of the enzyme [22]. Modelling of the protogen into the active site of tobacco PPO2 is based on the first model proposed by Koch et al. [22]. Residues Arg 98 , Phe 392 , Leu 372 and Leu 356 coordinated the inhibitor INH which was co-crystallised within the active site of PPO2. These four amino acid residues were found highly conserved throughout the PPO family. Ring A is coordinated via aromatic stacking with Phe 392 (Fig. 2 A) [22]. The side chains of Leu 356 and Leu 372 interact hydrophobically with ring B. The side chain of residue Arg 98 forms a salt bridge to the propionate carboxyl group of ring C. Additionally ring C might be stabilised by flipping and subsequent stacking of the conserved residue Phe 353 . The dome conformation of the protogen is probably stabilised by the carbonyl oxygen of the highly conserved Gly 175 . Biochemical Journal Immediate Publication. Published on 30 Nov 2006 as manuscript BJ20061321 Copyright 2006 Biochemical Society

  who underlined the importance of a hydrophilic, favourably positively charged amino acid at position R59 in the human enzyme. Replacing Asn 67 by tryptophan Biochemical Journal Immediate Publication. Published on 30 Nov 2006 as manuscript BJ20061321

  Consequently, generated mutant tobacco PPO2 enzymes, the single as well as the double mutants, may serve as a model for further investigations towards the structural understanding of the disease causing situation in the human PPO. Breckau, D., Mahlitz, E., Sauerwald, A., Layer, G. and Jahn, D. (2003) Oxygendependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese. J. Biol. Chem. 278, 46625-46631 25 Layer, G., Grage, K., Teschner, T., Schunemann, V., Breckau, D., Masoumi, A., Jahn, M., Heathcote, P., Trautwein, A. X. and Jahn, D. (2005) Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe-4S] cluster and the two bound S-adenosyl-L-methionines. J. Biol. Chem. 280, 29038-29046

Fig. 1 :

 1 Fig. 1: Reaction scheme of PPO.

Fig. 2 :

 2 Fig. 2: Active site cavity of PPO2. (A) Active site of tobacco PPO2. Depicted are the investigated Arg 98 , Leu 356 , Leu 372 and Phe 392 .

Figure 1

 1 Figure 1

Biochemical Journal Immediate Publication. Published on 30 Nov 2006 as manuscript BJ20061321

Mustajoki, P. (1980) Variegate porphyria. Twelve years' experience in Finland. Q. J. Med. 49, 191-203

Dean, G. (1971) Screening tests for porphyria. Lancet. 1, 86-87

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (JA 470-7-3). Biochemical Journal Immediate Publication. Published on 30 Nov 2006 as manuscript BJ20061321 Copyright 2006 Biochemical Society

TABLES

Table 1: Kinetic parameters of wildtype and mutant PPO2. The Michaelis Menten constant K m , the k cat and the k cat /K m values for the N. tabacum PPO2 were determined from substrate velocity plots by measuring the constant velocity formation of proto from protogen over the substrate range from 1-25 µM. Values were determined by the computerised Lineveaver Burk iterative curve fitting (SigmaPlot 8.0 Enzyme Kinetics v1.1). The FAD content of wildtype and mutant PPO2 were determined spectroscopically. The FAD content was calculated using standard FAD (Sigma). The standard error of the shown results was between 5 and 10 %.


Dominik N Muller

Cosima Schmidt

Eduardo Barbosa-Sicard

Maren Wellner

Volkmar Gross

Hantz Hercule

Marija Markovic

Horst Honeck

Friedrich C Luft

Wolf-Hagen Schunck
email: schunck@mdc-berlin.de

Mouse Cyp4a isoforms: enzymatic properties, genderand strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation

Keywords: CYP, cytochrome P450, AA, arachidonic acid, HETE, hydroxyeicosatetraenoic acid, EET, epoxyeicosatrienoic acid, 20-COOH-AA, eicosatetraen-1,20 Spodoptera frugiperda, IgG, immunoglobulin G, HPLC, high performance liquid chromatography, RP, reverse phase, NP, normal phase, GC, gas chromatography, MS, mass spectrometry, BK channel, calcium-activated potassium channel, mTALH, thick ascending limb of Henle cytochrome P450, arachidonic acid, eicosapentaenoic acid, mouse, gender, kidney 2

come

INTRODUCTION

Cytochrome P450 (CYP 2 ) enzymes of the CYP4A subfamily are found in the liver, kidney, intestine, lung, heart and brain of various mammals including rat, rabbit, mouse, and man [START_REF] Okita | Cytochrome P450 4A fatty acid omega hydroxylases[END_REF]. They conduct ωhydroxylation of fatty acids and related compounds. This reaction facilitates the degradation of long-chain fatty acids by initiating their conversion to dicarboxylic acids, that are efficiently oxidized by peroxisomal ß-oxidation [START_REF] Johnson | Regulation of P450 4A expression by peroxisome proliferator activated receptors[END_REF]. With arachidonic acid (AA; 20:4 n-6), CYP4A-catalyzed ω-hydroxylation yields 20hydroxyeicosatetraenoic acid . 20-HETE plays an important role in the regulation of renal vascular and tubular function. Altered renal CYP4A expression is associated with hypertension [START_REF] Capdevila | Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase[END_REF][START_REF] Mcgiff | 20-HETE and the kidney: resolution of old problems and new beginnings[END_REF][START_REF] Roman | P-450 metabolites of arachidonic acid in the control of cardiovascular function[END_REF].

Urinary 20-HETE excretion is increased in patients with cirrhosis [START_REF] Mcgiff | 20-HETE and the kidney: resolution of old problems and new beginnings[END_REF][START_REF] Sacerdoti | Eicosanoid excretion in hepatic cirrhosis. Predominance of 20-HETE[END_REF] and endothelial dysfunction [START_REF] Ward | Urinary 20-hydroxyeicosatetraenoic acid is associated with endothelial dysfunction in humans[END_REF].

Moreover, a polymorphic CYP4A11 variant is associated with human hypertension [START_REF] Gainer | Functional variant of CYP4A11 20hydroxyeicosatetraenoic acid synthase is associated with essential hypertension[END_REF]. The mechanisms linking 20-HETE to blood pressure regulation are only partially understood. Both increased and decreased 20-HETE levels can be associated with high blood pressure probably reflecting the dual role of 20-HETE to induce vasoconstriction (pro-hypertensive) and to promote natriuresis (anti-hypertensive) [START_REF] Roman | P-450 metabolites of arachidonic acid in the control of cardiovascular function[END_REF].

The CYP4A subfamily members expressed in mouse kidney include Cyp4a10, Cyp4a12 and Cyp4a14 [START_REF] Henderson | Deduced amino acid sequence of a murine cytochrome P-450 Cyp4a protein: developmental and hormonal regulation in liver and kidney[END_REF][START_REF] Heng | A novel murine P-450 gene, Cyp4a14, is part of a cluster of Cyp4a and Cyp4b, but not of CYP4F, genes in mouse and humans[END_REF][START_REF] Holla | Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension[END_REF][START_REF] Honeck | Cytochrome P450-dependent renal arachidonic acid metabolism in desoxycorticosterone acetate-salt hypertensive mice[END_REF][START_REF] Stec | Distribution of cytochrome P-450 4A and 4F isoforms along the nephron in mice[END_REF]. Two Cyp4a12 genes exist, Cyp4a12a and Cyp4a12b, that result from a tandem 100 kb duplication within the Cyp4abx cluster on chromosome 4 [14]. We showed earlier that renal 20-HETE production is decreased in desoxycorticosterone acetate (DOCA)-salt hypertensive mice [START_REF] Honeck | Cytochrome P450-dependent renal arachidonic acid metabolism in desoxycorticosterone acetate-salt hypertensive mice[END_REF]. Bezafibrate restored 20-HETE production and improved renal hemodynamics. Similarly, fenofibrate induced 20-HETE and prevented angiotensin (Ang) II-induced hypertension [15]. In both models, the 20-HETE producing CYP isoform(s) down-regulated in hypertension and induced by fibrates was not unequivocally identified. Targeted Cyp4a14 gene disruption resulted in increased AA ω-hydroxylase activities and caused hypertension in male mice [START_REF] Holla | Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension[END_REF]. The mechanism seems to involve increased plasma androgen levels in the Cyp4a14 gene-disrupted mice followed by androgen-induced up-regulation of Cyp4a12 [START_REF] Holla | Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension[END_REF].

Cyp4a10 gene disruption results in salt-sensitive hypertension via affecting the regulation of the kidney epithelial sodium channel [START_REF] Nakagawa | Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel[END_REF].

We cloned and expressed the Cyp4a10, Cyp4a12a, Cyp4a12b, and Cyp4a14 cDNAs, determined the substrate specificity of the recombinant enzymes, and analyzed their strain-and gender-specific relationship to renal 20-HETE production. We tested whether or not the two Cyp4a12 gene copies differ in terms of renal expression and enzymatic properties. For substrates, we relied on the CYP4A test substrate lauric acid (LA) to prove the successful reconstitution of enzymatic activities, AA to analyze the 20-HETE production capacity of the isoforms, and the fish oil omega-3 polyunsaturated fatty acid (PUFA), eicosapentaenoic acid (EPA; 20:5 n-3), which can replace AA in CYP-dependent eicosanoid production. Secondary hypertension and susceptibility to target organ damage in mice is largely sex and strain dependent. For example, 129/Sv mice are more susceptible to DOCA-salt hypertension than following primer pairs (5'-3'): TCG GAA TTC GCA ATG AGT GTC TCT GCT CTA A/ CCG CTC GAG CGG TCA GTG GTG GTG GTG GTG GTG GAG CTT CTT GAG ATG TAG (forward/reverse primer for Cyp4a10 with 5`-added Eco RI-and Xho I-site, respectively), ACG CGT AAG CTG TTG TAT CAT GAG TGC / GCT AGC CAT TTG AGC TGT CTT GTT CTG (forward/reverse primer for Cyp4a12a and Cyp4a12b with 5`-added Mlu I-and Nhe I-site, respectively), and CGA CTC GAT CCA GAA ACT AC / ACA GGA CAC ATT GCA GAG AG (forward/reverse primer for Cyp4a14). The PCR products (about 1.6 kb) were cloned into the plasmid pCR2.1 (TOPO-TA-Cloning kit, Invitrogen) and at least three different clones per isoform were sequenced (Invitek GmbH). For cloning of Cyp4a12b, the RT-PCR product obtained with the Cyp4a12a/Cyp4a12b-specific primers was first digested with Avr II, which selectively cleaves the Cyp4a12a cDNA and thus excludes it from cloning.

Expression of CYP enzymes

Coexpression of the individual CYP isoforms with the human NADPH-CYP reductase (CPR) was performed as described previously [19] using a baculovirus/Sf9 insect cell system (for the details see Supplemental online data). The CYP concentrations were estimated by means of CO-difference spectra using an extinction coefficient of 91 mM -1 cm -1 [START_REF] Omura | The Carbon Monoxide-Binding Pigment of Liver Microsomes. I. Evidence for Its Hemoprotein Nature[END_REF]. CPR activities were assayed in 50 mM Tris-HCL buffer, pH 7.5, containing 0.1 mM EDTA, 0.05 mM cytochrome c, 0.1 mM NADPH, and 2.2 mM KCN at 25°C using an extinction coefficient of 21 mM -1 cm -1 at 550 nm. The microsomes used for kinetic analysis had molar CYP:CPR ratios in the range of 1:0.9 to 1:1.2. For calculation, we assumed that 4.5 U (µmol cytochrome c/min) correspond to 1 nmol CPR based on the specific activity of the purified 79 kDa enzyme (60 U/mg).

Immunoblotting

Microsomal protein was separated on 10% SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Life Sciences). An antibody raised in goat against purified rat CYP4A1 (Daiichi Pure Chemicals Co., Ltd.) and a peptide-specific antibody raised in rabbits against mouse Cyp4a12a were used as primary antibodies. The antigenic peptide (NH3-SRRIQLQDEEELEKLKKKRR-COOH) corresponded to amino acids 266-286 of the Cyp4a12a protein (peptide synthesis by BIOSYNTHAN GmbH, immunization by BioGenes GmbH). Anti-goat IgG and anti-rabbit IgG peroxidase conjugate (Sigma Chemical Co) were used as secondary antibodies. Blots were developed with the chemiluminescence substrate from Boehringer-Mannheim and evaluated with the Image Reader LAS-1000 (Fuji Photo Film Co). Ponceau staining was used for verifying equal protein loading.

Fatty acid metabolism

[1-14 C]LA (2.11 GBq/mmol, Amersham Bioscience), [1-14 C]AA (2.07 GBq/mmol, Amersham Bioscience) and [1-14 C]EPA (2.0 GBq/mmol, Perkin Elmer Inc.) were used as substrates. Standard reactions with the recombinant Cyp4a isoforms were performed in 100 µl 100 mM potassium phosphate 5 buffer, pH 7.2 containing 10 pmol Cyp and the substrate at a concentration of 10 µM (AA and EPA) or 30 µM (LA). Reactions were started with NADPH (1 mM final concentration) after preincubating the microsomes with the substrates for 10 min at 37 °C. Reactions were terminated adding citric acid (20 mM final concentration). Reaction products were extracted into ethyl acetate, evaporated under nitrogen, and resuspended in ethanol. Incubations with cytochrome b5 (cytb5; Calbiochem) were done in a molar Cyp:cytb5 ratio of 1:1. Microsomal enzymes and cytb5 were preincubated in a total volume of 15 µl for 10 min on ice. For kinetic analysis, standard reactions were performed with seven different substrate concentrations between 1.25 and 50 µM and the experiments were done in triplicate. Reactions were terminated after 1 min (LA) or 3 min (AA and EPA). For analysis of regio-and stereoselectivities, standard reactions were up-scaled (4-to 10-fold; incubation times up to 10 min). With mouse renal microsomes, the standard reaction conditions described above were modified. The 100 mM potassium phosphate buffer, pH 7.2 was supplemented with 1 mM EDTA and 50 nM FAD and FMN and reactions were performed using 800 µg/ml renal microsomal protein with 40 µM AA for 30 min or with 200 µM LA for 3 min.

Soluble epoxide hydrolase assay

Soluble epoxide hydrolase activities were determined using the 10,000 x g supernatants obtained during the preparation of renal microsomes as described above. Reactions were performed at 37 °C for 10 min in 100 µl 100 mM potassium phosphate buffer, pH 7.2 containing 50 µM [1-14 C]-14,15epoxyeicosatetraenoic acid (EET). [1-14 C]-14,15-EET was prepared by chemical oxidation of radiolabeled AA according to [START_REF] Falck | Synthesis of epoxyeicosatrienoic acids and heteroatom analogs[END_REF]. The reactions were started by adding the 10,000 x g supernatant (5 µg of protein), and extracted as described above.

Analysis of metabolites

Metabolites were analyzed using the HPLC system LC-10Avp from Shimadzu equipped with a radioactivity monitor (LB509, Berthold). Total metabolites were resolved by reversed-phase (RP)-HPLC on a Nucleosil 100-5C18 HD column (250 x 4 mm; Macherey-Nagel). LA metabolites were esterified with diazomethane and resolved in RP-HPLC with a linear gradient of acetonitrile/water/acetic acid (29.5/70.5/0.1, v/v/v) to acetonitrile/water/acetic acid (59.5/40.5/0.1, v/v/v) over 30 min followed by 15 min acetonitrile/acetic acid (100/0.1, v/v) at a flow rate of 1 ml/min. AA and EPA metabolites were resolved with a linear gradient of acetonitrile/water/acetic acid (50/50/0.1, v/v/v) to acetonitrile/acetic acid (100/0.1, v/v) over 40 min at a flow rate of 1 ml/min. 19-and 20-HETE as well as 19-and 20hydroxyeicosapentaenoic acid (19/20-HEPE) were resolved by normal-phase (NP)-HPLC on a Nucleosil 100-5 column (250 x 4 mm; Macherey-Nagel) utilizing a linear gradient from hexane/2-propanol/acetic acid (99/1/0.1, v/v/v) to hexane/2-propanol/acetic acid (98.3/1.7/0.1, v/v/v) over 40 min at a flow rate of 1 ml/min as described [START_REF] Lauterbach | Cytochrome P450-dependent eicosapentaenoic acid metabolites are novel BK channel activators[END_REF]. 16-, 17-, and 18-HETE were resolved with hexane/2-propanol/ acetic acid 6 (100/0.4/0.1, v/v/v) at a flow rate of 1.5 ml/min according to [START_REF] Falck | Cytochrome P-450-dependent oxidation of arachidonic acid to 16-, 17-, and 18-hydroxyeicosatetraenoic acids[END_REF]. 17,18-epoxyeicosatetraenoic acid (17,18-EETeTr) was treated with diazomethane and the methyl ester was then resolved into the R,S and S,R enantiomers on a Chiracel OB column (250 x 4.6 mm; Daicell) using a linear gradient from hexane/2propanol (99.7/0.3, v/v) to hexane/2-propanol (98/2 v/v) over 40 min at a flow rate of 1 ml/min [START_REF] Barbosa-Sicard | Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily[END_REF].

Preparation of authentic LA [START_REF] Ortiz De Montellano | Specific inactivation of hepatic fatty acid hydroxylases by acetylenic fatty acids[END_REF], AA [START_REF] Schwarz | Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R),18(S)epoxyeicosatetraenoic acid[END_REF][START_REF] Carroll | Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells[END_REF] and EPA [START_REF] Barbosa-Sicard | Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily[END_REF][START_REF] Schwarz | Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R),18(S)epoxyeicosatetraenoic acid[END_REF][START_REF] Capdevila | The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3[END_REF] metabolites was performed as described in Supplemental online data.

Statistical Analysis

All data derived from the animal experiments are presented as means±SEM and were analyzed by ANOVA followed by the Tukey-Kramer multiple comparison test (InStat software; GraphPad Software Inc). The significance level was taken at P<0.05. Apparent K m and V max values of the recombinant Cyp4a enzymes were calculated using the "Enzyme Kinetics Module" of SigmaPlot 7 (SPSS).

RESULTS

Cloning of four different Cyp4a cDNAs

Cyp4a10: The Cyp4a10 cDNA cloned from NMRI showed 100 % identity to the corresponding mRNA (AK002528) and genomic sequences (contig NT_039264) from C57BL/6 mice. However, it differed in two positions from the Cyp4a10 mRNA of FVB/N mice (NM_010011), which has been used as the provisional NCBI reference sequence for Cyp4a10: 190A>C and 207A>C (counted from the start ATG), resulting in Q64K and Q69H substitutions (NMRI and C57BL/6 vs. FVB/N). Cyp4a12a: The Cyp4a12-specific primer pair yielded cDNA clones from renal RNA of both NMRI and C57BL/6, whose sequences completely agreed with that of the MGC25972 gene (C57BL/6 contig NT_039264). The encoded Cyp isoform was termed "Cyp4a10-like" or "similar to P450 4A8" on contig NT_039264 but will be designated in the following as Cyp4a12a according to [14]. cDNA sequences were so far only available for FVB/N mice (BC014721, NM_177406) and differ from that in NMRI and C57BL/6 by one nonsynonymous (391A>C; M131L) and one silent nucleotide substitution (1233C>G). Cyp4a12b: The gene originally termed "Cyp4a12" in the C57BL/6 genome (contig NT_039264) is located approximately 112 kbp downstream of Cyp4a12a. It will be designated here as Cyp4a12b according to [14]. The coding sequences of Cyp4a12a and Cyp4a12b differ at only 23 nucleotide positions resulting in 11 amino acid substitutions. The primer pair used in the present study allowed the amplification of both the Cyp4a12a and Cyp4a12b sequences. To enrich the Cyp4a12b cDNA, the RT-PCR product was digested with Avr II, which selectively cleaves the Cyp4a12a cDNA. In this way, clones were generated from C57BL/6 liver mRNA, which showed 100 % sequence identity to the predicted coding sequence of the Cyp4a12b gene ("Cyp4a12" on contig NT_039264). However, compared to the provisional NCBI reference sequence for the "Cyp4a12" mRNA (NM_172306, strain not specified), there is a nonsynonymous 494G>A substitution (V165H in contig NT_039264 and own cDNA sequence). Cyp4a14: The Cyp4a14 cDNA cloned from NMRI mice showed with one exception (13T>G resulting in L5V) 100 % identity to the coding region of the mRNA reference sequence NM_007822 (strain C57BL/6

x CBA) and to that predicted from the C57BL/6 genome (contig NT_039264). This polymorphism has been also observed in other mouse strains (rs3022990).

Metabolism of lauric acid by recombinant mouse Cyp4a isoforms

Co-expression of the individual Cyp4a isoforms with CPR in Sf9 insect cells yielded highly active microsomal enzyme systems that hydroxylated LA with V max values between 40 and 76 nmol/nmol/min and apparent K m values between 2 and 16 µM (Table 1). Addition of cytb5 in a 1:1 molar ratio doubled the V max values of Cyp4a10, Cyp4a12a and Cyp4a12b. In contrast, the activity of Cyp4a14 was not significantly influenced by cytb5 (data not shown).

The primary products of Cyp4a-catalyzed LA hydroxylation were 11-and 12-OH-LA. The identity of these metabolites was confirmed by co-migration with the authentic standard compounds (for representative HPLC chromatogramms see supplementary Fig. S1) and by GC-MS analysis as described in Supplemental online data. The 12-OH:11-OH ratio was isoform-specific (Table 1). The regioselectivity was very high with Cyp4a10 (94:6), moderate with Cyp4a12a (83:17) and Cyp4a14 (62:38) and almost not expressed with Cyp4a12b (48:52). Prolonged incubation or increased enzyme concentrations yielded substantial amounts of dodecan-1,12-dioic acid (DDDA) as a secondary product resulting from further oxidation of 12-OH-LA (data not shown).

Metabolism of arachidonic acid by recombinant mouse Cyp4a isoforms

AA was efficiently metabolized only by Cyp4a12a and Cyp4a12b (Table 2; for representative HPLC chromatogramms see supplementary Fig. S2). Major primary metabolites were 20-HETE and 19-HETE which were produced by both Cyp4a12a and Cyp4a12b in a ratio of about 90:10. Cyp4a12b produced another primary metabolite that co-migrated with 18-HETE in NP-HPLC and represented about 12 % of the total product. Secondary product formation analogous to the oxidation of LA to DDDA was also observed with AA. The secondary metabolite co-migrated in RP-HPLC with authentic eicosatetraen-1,20dioic acid (20-COOH-AA). The same metabolite was also directly produced from [1-14 C]20-HETE by the Cyp4a12 isoforms (supplementary Figs. S2B andS2H).

Michaelis-Menton kinetics were observed determining the AA conversion rates at different substrate concentrations. The apparent K m values were 25 and 43 µM for Cyp4a12a and Cyp4a12b, respectively. Both enzymes reached high V max values of about 10 nmol/min/nmol. The V max values were almost doubled in the presence of stoichiometric amounts of cytb5 (Table 2). With Cyp4a10 and Cyp4a14, AA metabolites were clearly detectable only after increasing the enzyme concentration to 500 nM and prolonging the reaction time (Table 2 and supplementary Figs. S2A andS2D). The metabolites observed with Cyp4a10 co-migrated with 19/20-HETE. Cyp4a14 was almost completely inactive as an AA hydroxylase, but produced small amounts of 11,12-EET. At a substrate concentration of 10 µM, Cyp4a10 reached an activity of 0.05 nmol/nmol/min (in the presence of cytb5) and was thus 70-fold less active than Cyp4a12a. At higher AA concentrations Cyp4a10 showed specific activities of 0.14 (40 µM), 0.21 (100 µM), and 0.63 nmol/nmol/min (200 µM), without reaching substrate saturation. Due to these low conversion rates, a further detailed kinetic analysis was not possible.

Metabolism of eicosapentaenoic acid by recombinant mouse Cyp4a isoforms

EPA was converted by Cyp4a12a and Cyp4a12b with high catalytic efficiencies that even slightly exceeded those determined for AA (Tab. 3). In comparison, Cyp4a10 and Cyp4a14 showed only a weak capacity to convert EPA resembling their very low activities in AA conversion (Table 3; for representative HPLC chromatogramms see supplementary Fig. S3).

Whereas Cyp4a12a and Cyp4a12b functioned solely as hydroxylases when converting AA, they catalyzed both a hydroxylation and an epoxidation reaction with EPA. The ratio of hydroxylase to epoxygenase activities was 1:1.3 with Cyp4a12a and 1:2 with Cyp4a12b. Accordingly, the epoxidation product represented the major EPA metabolite with both isoforms. The epoxidation product co-migrated in RP-HPLC with authentic 17,18-EETeTr. Chiral-phase HPLC resolved the 17,18-EETeTr peak collected from RP-HPLC into the respective R,S-and S,R-enatiomers. Cyp4a12a produced preferentially the R,S-enantiomer, whereas Cyp4a12b displayed no stereoselectivity (Table 3). The hydroxylase product consisted of 19-and 20-HEPE and both enzymes showed a regioselectivity in favor of producing the 20-OH metabolite (Table 3).

Gender-and strain-specific renal expression of mouse Cyp4a isoform

TaqMan-PCR analysis demonstrated that Cyp4a10 is highly expressed in the kidneys of both genders, whereas Cyp4a12a is predominantly a male-and Cyp4a14 a female-specific isoform (Fig. 1A-D).

Among the isoforms efficiently producing 20-HETE, only Cyp4a12a but not Cyp4a12b showed significant renal expression. Cyp4a12a levels in the males largely exceeded that in the females (45-fold for NMRI, 88-fold for FVB/N, 40-fold for 129 Sv/J, 7-fold for BALB/c and 48-fold for C57BL/6; Fig. 1B). Moreover, we observed marked differences in the renal Cyp4a12a mRNA levels between the five different male strains (Fig. 1B). The highest expression was observed in NMRI followed by FVB/N, BALB/c and 129 Sv/J. The renal Cyp4a12a expression level in male C57BL/6 reached only about 17 % of male NMRI and was statistically significantly lower compared to all other male strains. In contrast, Cyp4a12b mRNA was almost undetectable in the kidneys of both gender of all strains tested. Reliable TaqMan-PCR amplification curves were only obtained with renal samples from female 129 Sv/J, and male and female C57BL/6 (Fig. 1C). Renal samples from all other strains yielded low unspecific signals not different from the non-template controls. However, Cyp4a12b mRNA was easily detectable under the same experimental conditions using lung instead of renal samples (data not shown) indicating that Cyp4a12b shows a different tissue specificity of expression.

We also analyzed the gender and strain differences in Cyp4a isoform expression at the protein level. The antibody raised against a Cyp4a12a-derived peptide efficiently recognized recombinant Cyp4a12a and Cyp4a12b, showed a very weak cross-reactivity with Cyp4a10 and did not react with Cyp4a14 (Fig. 1E). In contrast, the CYP4A1-antibody detected all four mouse Cyp4a isoforms (Fig. 1E).

Western blot analysis of mouse renal microsomes confirmed the gender differences in Cyp4a12 isoform expression (Fig. 1F). The Cyp4a12-specific antibody yielded a single strong band with male and almost no reaction with female renal microsomes with exception of a very weak but reproducible Cyp4a12 protein expression in female 129 Sv/J and BALB/c (Fig. 1F).

Gender-and strain-specific renal microsomal arachidonic acid hydroxylase activities

Mouse renal microsomes of all strains tested showed both AA hydroxylase and AA epoxygenase activities (Fig. 2A). Renal microsomes of female mice had low AA hydroxylase activities ranging between 15 and 25 pmol/min/mg. Males showed 3 to 5-times higher activities than the respective female strain (Fig. 2B). The gender difference was most pronounced in NMRI, where males reached a renal AA hydroxylase activity of 93±4 compared to 20±0.5 pmol/min/mg in females. No significant genderdifference was observed in renal microsomal 20-HETE production of C57BL/6 (25±1 vs. 16±1 pmol/min/mg, male vs. female). Clear gender differences were also found in renal microsomal AA epoxygenase and in soluble epoxide hydrolase activities (Figs. 2C and2D). Compared to the large differences in 20-HETE production among male strains (Fig. 2B), strain differences in epoxygenase and hydrolase activities were less pronounced.

Induction of Cyp4a12a and of renal microsomal 20-HETE production by androgen treatment

Androgen treatment induced total renal and renal arteriole Cyp4a12a mRNA expression more than 4-fold in C57BL/6 and about 1.4-fold in NMRI (Figs. 3A and3B). Western blot analysis showed that the Cyp4a12 protein level was significantly higher in male NMRI compared to male C57BL/6 (Fig. 3C). 5α-DHT strongly increased the Cyp4a12 protein level in C57BL/6 (to 600 % of the untreated control) and to a lesser extent also in NMRI (to 200 %). Renal microsomal AA hydroxylase activities were 23±4 pmol/min/mg in untreated C57BL/6 and 128±14 pmol/min/mg in untreated NMRI (Fig. 3D). 5α-DHTtreatment increased the activity to 90±7 in C57BL/6 and to 167±11 pmol/min/mg in NMRI. These data show that 5α-DHT treatment significantly induced Cyp4a12a mRNA, protein and enzyme activity level in C57BL/6 leading to Cyp4a12a expression and 20-HETE production similar to NMRI already under basal conditions.

DISCUSSION

In this investigation, we identified Cyp4a12a as the predominant AA ω-hydroxylase in the mouse kidney and demonstrated that Cyp4a12a expression levels determine the sex and strain-specific differences in mouse renal 20-HETE production. We suggest the differences in 20-HETE we identified amongst the strains could account for resistance to target organ damage exhibited by some strains, but not others.

Furthermore, the sex differences we identified, coupled with the androgen effects we observed, may explain the relative resistance of female mice to target organ damage compared to males within strains.

Recombinant Cyp4a12a showed a high regioselectivity in favor of producing 20-HETE from AA similar as observed before with mouse renal microsomes [START_REF] Honeck | Cytochrome P450-dependent renal arachidonic acid metabolism in desoxycorticosterone acetate-salt hypertensive mice[END_REF]. Cyp4a12a did not form any detectable amounts of AA epoxides. However with EPA, Cyp4a12a efficiently catalyzed the epoxidation of the 17,18-double bond that distinguishes this n-3 PUFA from AA. Thus, Cyp4a12a generates very different sets of metabolites when either AA or EPA becomes accessible as substrate. Such a shift in CYPdependent eicosanoids may have physiological significance. 20-HETE is a powerful endogenous vasoconstrictor that functions via inhibition of BK channels in vascular smooth muscle cells [START_REF] Mcgiff | 20-HETE and the kidney: resolution of old problems and new beginnings[END_REF]. In contrast, 17,18-EETeTr shows vasodilator properties and is a very potent activator of BK channels [START_REF] Lauterbach | Cytochrome P450-dependent eicosapentaenoic acid metabolites are novel BK channel activators[END_REF]29]. BK channel activation is highly stereoselective [START_REF] Lauterbach | Cytochrome P450-dependent eicosapentaenoic acid metabolites are novel BK channel activators[END_REF] and we found that Cyp4a12a produces preferentially the active R,S-enantiomer. Alterations in cyclooxygenase-and lipoxygenase-dependent prostanoid and leukotriene biosynthesis have been generally assumed to contribute to the beneficial effects of fish oil n-3 PUFAs in cardiovascular diseases [30]. Considering the enzymatic properties of Cyp4a12a described above, it appears highly probable that also CYP-catalyzed 20-HETE production can be manipulated under in vivo conditions by n-3 PUFA-rich diets. How this state-of-affairs affects 20-HETE-mediated signaling pathways regulating vascular and renal function will be an important research topic.

Cyp4a12a did not only hydroxylate AA to 20-HETE but also catalyzed the subsequent oxidation steps necessary to convert 20-HETE to 20-COOH-AA. The ability to catalyze an oxidation cascade from a given fatty acid to the corresponding dicarboxylic acid was shown earlier for several CYP enzymes, examining other substrates than AA [START_REF] Scheller | Oxygenation cascade in conversion of n-alkanes to alpha,omega-dioic acids catalyzed by cytochrome P450 52A3[END_REF]. Alcohol dehydrogenase 4 may be the responsible enzyme in vascular smooth muscle and endothelial cells that converts 20-HETE to 20-COOH-AA [START_REF] Collins | Omega-oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4[END_REF]. This reaction may serve to overcome the vasoconstrictor action of 20-HETE [START_REF] Carroll | Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells[END_REF][START_REF] Kaduce | 20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells[END_REF]. Moreover, 20-COOH-AA is known to inhibit the Na,K,2Cl-cotransporter in mTALH [START_REF] Mcgiff | 20-HETE and the kidney: resolution of old problems and new beginnings[END_REF][START_REF] Carroll | Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells[END_REF] and to function as a dual activator of peroxisome proliferator-activated receptors α and γ [START_REF] Fang | 20-Caboxy-arachidonic acid is a dual activtor of peroxisome proliferator-activated receptors α and γ[END_REF].

Our study indicates that both Cyp4a12 genes [14] are functional but differ significantly in the tissue specificity of expression. Considering the very high degree of homology between the two Cyp4a12 variants, the differences are remarkable that we observed comparing their enzymatic properties. In contrast to Cyp4a12a, Cyp4a12b showed (i) no preference for ωover ω-1 hydroxylation when converting LA, (ii) no stereoselectivity when epoxidizing the 17,18-double bond of EPA, and (iii) a higher EPA epoxygenase:EPA hydroxylase ratio. These findings indicate that the positioning of the substrate alkyl chain is probably less strict in the active site of Cyp4a12b compared to that in Cyp4a12a.

Interestingly, there are also similar pairs of highly homologous CYP4A enzymes in man (CYP4A11 and CYP4A22; [START_REF] Gainer | Functional variant of CYP4A11 20hydroxyeicosatetraenoic acid synthase is associated with essential hypertension[END_REF]35]) and rat (CYP4A2 and CYP4A3; [START_REF] Nguyen | Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoformspecific inhibitors[END_REF][START_REF] Hoch | Molecular basis for the omegaregiospecificity of the CYP4A2 and CYP4A3 fatty acid hydroxylases[END_REF]). However, the sequence differences in these CYP pairs concern other amino acid positions and have different functional consequences. Thus, the significance of the CYP4A gene duplications and of the subsequently evolved gene variants is not readily obvious and may differ in human, rat and mouse.

Cyp4a10 is the murine Cyp isoform showing the highest homology to CYP4A1 (92 % amino acid sequence identity), the most active rat AA hydroxylase [START_REF] Nguyen | Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoformspecific inhibitors[END_REF]. However, our data show that Cyp4a10 displays only a very weak AA hydroxylase activity. Assuming that 10 µM is a reasonable physiological AA concentration, the activity of Cyp4a10 would be about 70-fold lower than that of Cyp4a12a. Cyp4a10 gene disruption did not alter mouse renal AA hydroxylase activities confirming that the Cyp4a10 enzyme does not contribute to 20-HETE production [START_REF] Nakagawa | Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel[END_REF]. Cyp4a12a and Cyp4a12b have 78 % sequence identity to CYP4A1 and are most closely related to CYP4A8 (87 %) which showed, however, the lowest AA hydroxylase activity among the rat CYP4A isoforms [START_REF] Nguyen | Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoformspecific inhibitors[END_REF]. CYP4A8 shares with Cyp4a12a the inducibility by androgens [START_REF] Nakagawa | Androgen-mediated induction of the kidney arachidonate hydroxylases is associated with the development of hypertension[END_REF]. Cyp4a14 is most similar to CYP4A2 (88 %) and CYP4A3 (89 %). The latter are highly expressed in the male rat kidney and display a significant 20-HETE synthase activity [START_REF] Nguyen | Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoformspecific inhibitors[END_REF]. In contrast, Cyp4a14 shows a female specific expression and we could not detect any 20-HETE synthase activity with this isoform.

Our data on the sex specificity of renal Cyp4a gene expression in five different mouse strains show in agreement with previous studies [START_REF] Heng | A novel murine P-450 gene, Cyp4a14, is part of a cluster of Cyp4a and Cyp4b, but not of CYP4F, genes in mouse and humans[END_REF][START_REF] Holla | Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension[END_REF] that Cyp4a10 is highly expressed in both sexes, whereas Cyp4a12a is clearly a male and Cyp4a14 a female specific isoform. Moreover, we found that the expression of Cyp4a12a is correlated to the capacity of mouse renal microsomes to hydroxylate AA thus providing further evidence for the role of this isoform in 20-HETE production. Comparing the males of the different strains, NMRI mice had clearly the highest Cyp4a12a expression levels whereas C57BL/6 appears as a naturally occurring mouse model with a specific knock-down in Cyp4a12a/20-HETE expression. Our results and those of other authors [START_REF] Holla | Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension[END_REF] demonstrate that Cyp4a12 is inducible by androgens. The androgen effect was much more pronounced with C57BL/6 compared to NMRI. Providing a possible explanation, mouse strains have been long known to differ in blood testosterone levels and C57BL/6 is considered as being chronically testosterone deficient [39]. The androgendependent expression also explains the low of Cyp4a12a levels in the females of all strains. We noticed, however, that the gender differences in Cyp4a12a mRNA and protein expression levels were apparently more pronounced than in the AA hydroxylase activities. Therefore, it remains to be analyzed whether the low but still significant renal microsomal AA hydroxylase activities in the females are in part due to different Cyp isoforms possibly including Cyp4F subfamily members [START_REF] Stec | Distribution of cytochrome P-450 4A and 4F isoforms along the nephron in mice[END_REF]40]. 20-HETE plays an important role in the regulation of various cardiovascular processes. It acts as a second messenger in signaling pathways modulating vascular tone, salt excretion, cell proliferation and angiogenesis [START_REF] Mcgiff | 20-HETE and the kidney: resolution of old problems and new beginnings[END_REF]. Since the intensity of these signaling pathways may depend on the extent of 20-HETE production, the question arises whether sex and strain-specific differences in the response to prohypertensive and pro-hypertrophic stimuli are partially due to differences in Cyp AA ω-hydroxylase expression levels. Substantiating this possibility, androgen-induced overproduction of 20-HETE in mice [START_REF] Holla | Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension[END_REF] and rats [START_REF] Nakagawa | Androgen-mediated induction of the kidney arachidonate hydroxylases is associated with the development of hypertension[END_REF] is associated with the development of hypertension. Moreover, rats transduced with an adenovirus for vascular overexpression of CYP4A2 developed endothelial dysfunction and hypertension [START_REF] Wang | Endothelial dysfunction and hypertension in rats transduced with CYP4A2 adenovirus[END_REF]. Female mice, which have very low renal 20-HETE production, are more resistant to Ang II-induced hypertension [START_REF] Xue | Sex differences in the development of angiotensin II-induced hypertension in conscious mice[END_REF] and to ischemia/reperfusion-induced kidney injury [42], compared with males. C57BL/6 males, which have lower renal 20-HETE production than the males of other strains, show reduced susceptibilities to the development of DOCA-salt hypertension [START_REF] Hartner | Strain differences in the development of hypertension and glomerular lesions induced by deoxycorticosterone acetate salt in mice[END_REF], glomerulosclerosis [43] and injury-induced neointimal hyperplasia [START_REF] Kuhel | Distinction in genetic determinants for injury-induced neointimal hyperplasia and diet-induced atherosclerosis in inbred mice[END_REF]. Recently, CYP-dependent AA metabolism has been also implicated in cardiac function and ischemia/reperfusion-induced myocardial injury [45]. Clearly, there are many other factors potentially involved in mediating gender-related differences in the manifestation of cardiovascular disease [46] and the relative importance of differences in CYP-dependent AA metabolism remains to be determined.

Our study provides comprehensive information on the expression and enzymatic properties of the Cyp4a subfamily members in the mouse kidney, identifies Cyp4a12a as the predominant 20-HETE synthase, and suggests that differences in Cyp4a12a/20-HETE expression may be involved in determining sex and strain-specific susceptibility differences to hypertension and other cardiovascular diseases.

Transgenic and gene-deletion approaches to alter Cyp4a12a expression in selected tissues may help to directly prove the various physiological and pathophysiological functions attributed to 20-HETE in the regulation of renal, vascular, and cardiac function. In those studies, strain and sex selection will be a highly important aspect of study design. Significant differences (p<0.05) between the groups are marked as follows: * vs. untreated C57BL/6; # vs. untreated NMRI; + vs. testosterone treated C57BL/6.

FIGURE 1 .

 1 FIGURE 1. Gender-and strain-specific renal expression of Cyp4a-isoforms (A-D):Renal mRNA expression of Cyp4a10, Cyp4a12a, Cyp4a12b, and Cyp4a14 in male and female NMRI, FVB/N, 129 Sv/J, BALB/c, and C57BL/6 mice. Data are mean±SEM (n=5 in each group). Significant differences (p<0.05) between the groups are marked as follows: * vs. other gender of the same strain; # vs. other males; + vs. other females; § vs. 129 Sv/J, BALB/c and C57BL/6; ^ vs. 129 Sv/J and C57BL/6; ° vs. NMRI. (E): Specificity of the antibodies used for Western blot analysis. Control -microsomal protein (8 µg) isolated from Sf9 cells not expressing any of the Cyp isoforms; other lanes -individual recombinant Cyp4a isoforms (0.2 pmoles each); ab: 4A1 -antibody raised against rat CYP4A1; ab: 4a12 -antibody raised against a Cyp4a12aspecific peptide. (F): Western blot analysis of renal Cyp4a protein expression using the primary 4A1 and 4a12 antibodies; 7,5 µg total microsomal protein were loaded per lane.

FIGURE 2 .

 2 FIGURE 2. Gender-and strain-specific expression of enzymatic activities involved in mouse renal arachidonic acid metabolism. (A): Representative RP-HPLC chromatogram of AA metabolites produced by renal microsomes isolated from male and female NMRI mice. (B-D): Renal AA hydroxylase, AA epoxygenase, and soluble epoxide hydrolase activities of male and female NMRI, FVB/N, 129 Sv/J, BALB/c, and C57BL/6. Data are mean±SEM (n=6 in each group). Significant differences (p<0.05) between the groups are marked as follows: * vs. other gender of the same strain; # vs. other males; + vs. FVB/N and C57BL/6; § vs. BALB/c.

FIGURE 3 .

 3 FIGURE 3. Effect of 5α-dihydrotestosterone on Cyp4a12a expression and renal microsomal 20-HETE production in male NMRI and C57BL/6. (A, B): Cyp4a12a mRNA levels in total kidney and renal arterioles; (C): renal Cyp4a12 protein levels; (D): renal microsomal AA hydroxylase activities. Results are mean±SEM (n=6 each).

Biochemical Journal Immediate Publication. Published on 20 Nov 2006 as manuscript BJ20061328

ACKNOWLEDGEMENTS

The authors wish to thank Christel Andrée, Ramona Zummach and Ilona Kamer for excellent technical assistance. This study was supported by grants-in-aid to D.N. Muller, F.C. Luft and W.-H. Schunck from the Deutsche Forschungsgemeinschaft.

TABLES

The metabolites were resolved and quantified by RP-followed by NP-or chiral-phase HPLC as shown in supplementary Fig. S3. The values are averages from experiments with at least three different microsomal preparations. The standard errors were less than 10 % of the mean. b) Activities of Cyp4a10 and Cyp4a14 are given for a substrate concentration of 10 µM since a detailed kinetic analysis was not possible due to the very weak activities of these isoforms towards EPA. For comparison, under the same conditions, the corresponding values for Cyp4a12a were 3.6 and 6.7 (+ cytb5) and for Cyp4a12b 3.0 and 4.2 (+cytb5) nmol/nmol/min.