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.

Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms

“…It is clear that many additional P450 isoforms remain to be studied in the context of opioid analgesia. Among these, members of the CYP4X (Iliff et al, 2010) and CYP2J (Graves et al, 2013) subfamilies have epoxygenase activity and are found in the CNS. Although the P450 epoxygenase hypothesis has not been confirmed, new classes of analgesic or antihyperalgesic medications are already being developed to mimic the actions of lipid epoxides (Brostram and Falck, 2013) or to inhibit their metabolism (Wagner et al, 2013).…”

Quantitative Polymerase Chain Reaction Analysis of the MouseCyp2jSubfamily: Tissue Distribution and Regulation

“…Specific primer sets of oligonucleotides were designed, spanning exon/exon boundaries if possible, for the mouse Cyp2j subfamily members (Table 2). Some primer sets were those that had previously been published for use in nonquantitative RT-PCR Graves et al, 2013). All primers were diluted to a concentration of 10 mM, and 0.5 ml was used in a total volume of 10 ml for the qPCR reactions.…”

“…Mouse Cyp2j subfamily members are active in the metabolism of AA and other substrates Qu et al, 2001;Ma et al, 2002;Graves et al, 2013). The majority of P450 enzymes are abundantly expressed in the liver, but the mouse Cyp2j subfamily members are predominantly found in extrahepatic tissues.…”

“…The majority of P450 enzymes are abundantly expressed in the liver, but the mouse Cyp2j subfamily members are predominantly found in extrahepatic tissues. Previous studies indicated that Cyp2j5, Cyp2j8, Cyp2j11, and Cyp2j13 are predominantly expressed in the kidney Graves et al, 2013), Cyp2j6 transcripts are primarily detected in the intestine , and Cyp2j8, Cyp2j9, and Cyp2j12 transcripts are highly expressed in the brain (Qu et al, 2001;Graves et al, 2013). These previous studies have all used nonquantitative reverse-transcription polymerase chain reaction (RT-PCR) to detect the presence or absence of mouse Cyp2j transcripts in tissue.…”

“…Accurate qPCR of the mouse Cyp2j subfamily members will allow for assessment of their tissue distribution and aid in elucidating their biologic function and regulation. Developing specific primer sets for use with the SYBR Green reporter can be challenging due to nucleotide coding sequence homology (75%-88% identity) between the subfamily members (Graves et al, 2013).…”