mRNA distribution and heterologous expression of orphan cytochrome P450 20A1.
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.
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Katarina
K
Stark
Zhong-Liu
ZL
Wu
Cheryl J
CJ
Bartleson
F Peter
FP
Guengerich
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002784:Cholesterol / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018076:DNA, Complementary; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; C532758:CYP20A1 protein, human