Genomic Cloning and Protein Expression of a Novel Rat Brain Cytochrome P-450 CYP2D18* Catalyzing Imipramine N-Demethylation

Abstract

We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1Kawashima H. Strobel H.W. Biochem. Biophys. Res. Commun. 1995; 209: 535-540Crossref PubMed Scopus (42) Google Scholar). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5′-extension, and we posit in this report by analysis of a genomic clone that this 5′-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2669) Google Scholar). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine. We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1Kawashima H. Strobel H.W. Biochem. Biophys. Res. Commun. 1995; 209: 535-540Crossref PubMed Scopus (42) Google Scholar). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5′-extension, and we posit in this report by analysis of a genomic clone that this 5′-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2669) Google Scholar). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.