Identification of a novel cytochrome P450, CYP4X1, with unique localization specific to the brain,☆☆

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Abstract

A novel cytochrome P450 (P450 or CYP) isoform belonging to the CYP4 family was cloned with reverse transcription-polymerase chain reaction from rat brain. The nucleotide sequence contained an open reading frame coding for 507 amino acids. The deduced amino acid sequence showed 41–51% identity with that of members of the rat CYP4 subfamilies 4A, 4B, and 4F. The enzyme was designated CYP4X1. Northern blot analysis showed that CYP4X1 is highly and specifically expressed in the brain. In situ hybridization experiments suggest that CYP4X1 is mainly expressed in neurons in different regions, e.g., the brain stem, hippocampus, cortex, and cerebellum as well as in vascular endothelial cells. The function of this novel P450 enzyme is unknown, but the expression pattern of CYP4X1 suggests that it is possible that CYP4X1 plays a role in neurovascular function. The catalytic properties and physiological function of CYP4X1 are currently under investigation.

Section snippets

Materials and methods

Sequence analysis. Data on human CYP4X1 ESTs were obtained from the UniGene database [21]. The BLAST algorithm was used for searches of the HTGS sections in the GenBank [20]. Multiple alignments were prepared by Jotun Hein method of the Megaline program (DNA Star, Madison, WI). The genomic sequence used for analysis of the mouse cyp4x1 gene was a 234-kb long draft sequence (Accession No. AJ297131) [19] and the genomic sequences used for analysis of the human CYP4X1 gene were two 178-kb long

CYP4X1 cDNA

RT-PCR using primers designed toward two putative exons of a mouse cyp4x1 genomic sequence yielded a partial CYP4X1 cDNA sequence from rat brain, whereas no PCR bands were generated from either rat liver, kidney or cultured astrocytes. A 3-RACE method in combination with the usage of a 5-UTR specific primer for the mouse gene was used to obtain the sequence of the full coding region of the rat CYP4X1 cDNA. The nucleotide sequence contained an open reading frame of 1521-bp (Fig. 1). The 3

Discussion

In this study, we report the cDNA cloning and initial characterization of a rat brain specific P450 isoform belonging to a novel CYP4 subfamily. The deduced amino acid sequence of the enzyme exhibits 41–51% identity with that of members of the rat CYP4 subfamilies 4A, 4B, and 4F. According to the P450 nomenclature standard, this enzyme did not fulfill the criteria for belonging to any of the characterized mammalian subfamilies [1]. The enzyme was designated rat CYP4X1. It has been hypothesized

Acknowledgements

This work was supported by NIH/NINDS R01 NS32321-05, NIH/NHLBI P01 HL59996, VA Merit 3440-02P, and Wenner-Gren Foundations. We thank Dr. Pompon (CNRS, France) for the generous gift of the S. cerevisiae strain W(R).

References (28)

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Abbreviations: CYP, cytochrome P450; P450, cytochrome P450; EST, expressed sequence tag; HTGS, high throughput genomic sequences; RACE, rapid amplification of cDNA ends; RT-PCR, reverse trancription-polymerase chain reaction; UTR, untranslated region.

☆☆

The sequence reported in this paper has been deposited in the GenBank Accession No. AF439343.

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