Cloning ofCYP2J2Gene and Identification of Functional Polymorphisms

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Abstract
CYP2J2 is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that possess potent anti-inflammatory, vasodilatory, and fibrinolytic properties. We cloned and sequenced the entire CYP2J2 gene (∼40.3 kb), which contains nine exons and eight introns. We then sequenced theCYP2J2 exons and intron-exon boundaries in 72 healthy persons representing African, Asian, and European/white populations as part of the National Institutes of Health/National Institute of Environmental Health Sciences Environmental Genome Single Nucleotide Polymorphism Program. A variety of polymorphisms were found, four of which resulted in coding changes (Arg158Cys, Ile192Asn, Asp342Asn, and Asn404Tyr). A fifth variant (Thr143Ala) was identified by screening a human heart cDNA library. All five variant cDNAs of CYP2J2 were generated by site-directed mutagenesis and expressed in Sf9 insect cells by using a baculovirus system. The recombinant wild-type and variant CYP2J2 proteins immunoreacted with peptide-based antibodies to CYP2J2 and displayed typical cytochrome P450 (P450) CO-difference spectra; however, the Asn404Tyr and Ile192Asn variants also had prominent spectral peaks at 420 nm. The ability of these variants to metabolize arachidonic acid and linoleic acid was compared with that of wild-type CYP2J2. Three variants (Asn404Tyr, Arg158Cys, and Thr143Ala) showed significantly reduced metabolism of both arachidonic acid and linoleic acid. The Ile192Asn variant showed significantly reduced activity toward arachidonic acid only. The Asp342Asn variant showed similar metabolism to wild-type CYP2J2 for both endogenous substrates. Based on these data, we conclude that allelic variants of the humanCYP2J2 gene exist and that some of these variants result in a P450 protein that has reduced catalytic function. Insofar as CYP2J2 products have effects in the cardiovascular system, we speculate that these variants may be functionally relevant.