Abstract
Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver, and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3- to 4-fold higher in brain and heart than in liver. In situ hybridization of rat whole brain sections showed an mRNA expression pattern similar to that observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol of P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function.
Footnotes
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↵4 In situ hybridization of human tissues was attempted; however, no conclusions could be drawn from the limited number of analyses due to lack of material.
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This work was supported in part by the by Henning and Johan Trone Holst stiftelse (K.S.), Svenska Läkaresällskapet och Apotekarsocietéten (K.S.), and U.S. Public Health Service Grants R37 CA090426, T32 ES007028, and P30 ES000267 (F.P.G.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.022020.
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ABBREVIATIONS: P450, cytochrome P450 [also termed “heme thiolate P450” (Palmer and Reedijk, 1992); CYP indicates the gene in question]; bp, base pair(s); NCBI, National Center for Biotechnology Information; SSC, 15 mM sodium citrate buffer (pH 7.0) containing 150 mM NaCl); RT, reverse transcriptase; PCR, polymerase chain reaction; GAPDH, glycerylaldehyde 3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction; DIG, digoxigenin; TBS, Tris-buffered saline [0.10 M Tris-HCl (pH 7.5) containing 0.15 M NaCl]; UPLC, ultra-performance liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. -
↵1 Current affiliation: Experimental Asthma and Allergy Research, National Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden.
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↵2 Current affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, People's Republic of China.
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↵3 Current affiliation: CellzDirect Inc., Invitrogen Corporation, Austin, TX.
- Received April 23, 2008.
- Accepted June 4, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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