Characterization of four new mouse cytochrome P450 enzymes of the CYP2J subfamily

Abstract

The cytochrome P450 superfamily encompasses a diverse group of enzymes that catalyze the oxidation of various substrates. The mouse CYP2J subfamily includes members that have wide tissue distribution and are active in the metabolism of arachidonic acid (AA), linoleic acid (LA), and other lipids and xenobiotics. The mouse Cyp2j locus contains seven genes and three pseudogenes located in a contiguous 0.62 megabase cluster on chromosome 4. We describe four new mouse CYP2J isoforms (designated CYP2J8, CYP2J11, CYP2J12, and CYP2J13). The four cDNAs contain open reading frames that encode polypeptides with 62-84% identity with the three previously identified mouse CYP2Js. All four new CYP2J proteins were expressed in Sf21 insect cells. Each recombinant protein metabolized AA and LA to epoxides and hydroxy derivatives. Specific antibodies, mRNA probes, and polymerase chain reaction primer sets were developed for each mouse CYP2J to examine their tissue distribution. CYP2J8 transcripts were found in the kidney, liver, and brain, and protein expression was confirmed in the kidney and brain (neuropil). CYP2J11 transcripts were most abundant in the kidney and heart, with protein detected primarily in the kidney (proximal convoluted tubules), liver, and heart (cardiomyocytes). CYP2J12 transcripts were prominently present in the brain, and CYP2J13 transcripts were detected in multiple tissues, with the highest expression in the kidney. CYP2J12 and CYP2J13 protein expression could not be determined because the antibodies developed were not immunospecific. We conclude that the four new CYP2J isoforms might be involved in the metabolism of AA and LA to bioactive lipids in mouse hepatic and extrahepatic tissues.

Fig. 1.

Organization of the mouse Cyp2j subfamily on chromosome 4. Exon sequences of known mouse, rat, and human Cyp2j subfamily members were used to BLAST search the mouse genome in the Celera Discovery System and the National Center for Biotechnology Information databases. Seven Cyp2j genes (black arrows) and three pseudogenes (gray arrows) were mapped to the negative strand in a 0.62-Mb cluster on chromosome 4.

Fig. 2.

Amino acid sequences for the mouse CYP2J subfamily members. Conserved regions present in the deduced amino acid sequences of the CYP2Js are denoted as follows: the proline-rich region is underlined; the six substrate recognition sites (SRS) are bound in solid lines; the heme-binding region is bound in a hatched line with the invariant cysteine in bold and highlighted; and the location of the peptides used to raise polyclonal antibodies is highlighted. Stars indicate conserved amino acids among the seven CYP2J isoforms.

Fig. 3.

CO difference spectra of the new recombinant mouse CYP2J proteins. All the new CYP2J recombinant proteins have Soret maxima at approximately 450 nm. The scale for the absorbance for each of the CYP2Js for 0.001 OD (optical density) is as follows: CYP2J8 is 0.44 mm, CYP2J11 is 2.1 mm, CYP2J12 is 3.6 mm, and CYP2J13 is 1.9 mm.

Fig. 4.

Liquid chromatography–tandem spectrometry (LC-MS/MS) profiles for the metabolism of AA and LA by the recombinant CYPJs. A representative chromatogram of the incubation of CYP2J8 with AA and with (top) or without (bottom) NADPH is shown. Detection of individual AA metabolites is indicated. Experiments were performed in triplicate for each P450 enzyme.

Fig. 5.

Tissue distribution of the new CYP2J transcripts by RT-PCR. Total RNA extracted from multiple mouse tissues was analyzed by a one-step RT-PCR with sequence-specific primer pairs (Table 3). All tissues, unless specified or female-specific, were from males. Primer specificity was determined using the mouse CYP2J cDNAs as templates. A mouse β-actin primer pair was used as a control for RNA quantity. The results are representative of three independent experiments.

Fig. 6.

Specificity and tissue distribution of the new CYP2Js by immunoblotting. (A) CYP2J recombinant proteins isolated from Sf21 insect cells infected with baculovirus stocks (0.5 pmol of P450/lane) were immunoblotted with α-CYP2J8pep1, α-CYP2J11pep1, α-CYP2J12pep1, or α-CYP2J13pep1. (B) Total tissue lysates (50 μg protein/lane) were prepared from various mouse tissues and immunoblotted with α-CYP2J8pep1. Microsomal fractions (40 μg protein/lane) prepared from various mouse tissues were immunoblotted with α-CYP2J11pep1. The results are representative of three independent experiments.

Fig. 7.

Immunohistochemical staining of CYP2J8 protein in the brain and CYP2J11 protein in the heart and kidney. Sections of the brain were immunostained with (A) α-CYP2J8pep1 or (B) normal rabbit serum as described in Materials and Methods. Diffuse staining for CYP2J8 was present in the neuropil of the gray matter of the brain with no staining of the white matter. Sections of (C and D) heart and (E and F) kidney were immunostained with (C and E) α-CYP2J11pep1 or (D and F) normal rabbit serum. Staining for CYP2J11 protein was present in cardiomyocytes and renal proximal tubules.