CYP2U1, a Novel Human Thymus- and Brain-specific Cytochrome P450, Catalyzes ω- and (ω-1)-Hydroxylation of Fatty Acids

Long chain fatty acids have recently emerged as critical signaling molecules in neuronal, cardiovascular, and renal processes, yet little is presently known about the precise mechanisms controlling their tissue distribution and bioactivation. We have identified a novel cytochrome P450, CYP2U1, which may play an important role in modulating the arachidonic acid signaling pathway. Northern blot and real-time PCR analysis demonstrated that CYP2U1 transcripts were most abundant in the thymus and the brain (cerebellum), indicating a specific physiological role for CYP2U1 in these tissues. Recombinant human CYP2U1 protein, expressed in baculovirus-infected Sf9 insect cells, was found to metabolize arachidonic acid exclusively to two region-specific products as determined by liquid chromatography-mass spectrometry. These metabolites were identified as 19- and 20-hydroxy-modified arachidonic acids by liquid chromatography-tandem mass spectrometry analysis. In addition to ω/ω-1 hydroxylation of arachidonic acid, CYP2U1 protein also catalyzed the hydroxylation of structurally related long chain fatty acid (docosahexaenoic acid) but not fatty acids such as lauric acid or linoleic acid. This is the first report of the cloning and functional expression of a new human member of P450 family 2, CYP2U1, which metabolizes long chain fatty acids. Based on the ability of CYP2U1 to generate bioactive eicosanoid derivatives, we postulate that CYP2U1 plays an important physiological role in fatty acid signaling processes in both cerebellum and thymus. Long chain fatty acids have recently emerged as critical signaling molecules in neuronal, cardiovascular, and renal processes, yet little is presently known about the precise mechanisms controlling their tissue distribution and bioactivation. We have identified a novel cytochrome P450, CYP2U1, which may play an important role in modulating the arachidonic acid signaling pathway. Northern blot and real-time PCR analysis demonstrated that CYP2U1 transcripts were most abundant in the thymus and the brain (cerebellum), indicating a specific physiological role for CYP2U1 in these tissues. Recombinant human CYP2U1 protein, expressed in baculovirus-infected Sf9 insect cells, was found to metabolize arachidonic acid exclusively to two region-specific products as determined by liquid chromatography-mass spectrometry. These metabolites were identified as 19- and 20-hydroxy-modified arachidonic acids by liquid chromatography-tandem mass spectrometry analysis. In addition to ω/ω-1 hydroxylation of arachidonic acid, CYP2U1 protein also catalyzed the hydroxylation of structurally related long chain fatty acid (docosahexaenoic acid) but not fatty acids such as lauric acid or linoleic acid. This is the first report of the cloning and functional expression of a new human member of P450 family 2, CYP2U1, which metabolizes long chain fatty acids. Based on the ability of CYP2U1 to generate bioactive eicosanoid derivatives, we postulate that CYP2U1 plays an important physiological role in fatty acid signaling processes in both cerebellum and thymus. Members of the cytochrome P450 (P450) 1The abbreviations used are: P450, cytochrome P450; AA, arachidonic acid; DHA, docosahexaenoic acid; DTA, docosatraenoic acid; EET, epoxy-modified arachidonic acid; EPA, eicosapentaenoic acid; EST, expressed sequence tag; ETA, eicosatrienoic acid; hb5, human cytochrome b5; HETE, hydroxy-modified arachidonic acid; hOR, human cytochrome P450-NADPH reductase; HPETE, hydroperoxide; HPLC, high performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; 17-ODYA, 17-octodecynoic acid. family of enzymes play key roles in tissue-specific conversion of natural substrates into locally active hormones, vitamins, and signaling molecules including derivatives of arachidonic acid (AA) known as eicosanoids (1Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar). This diverse group of substrates is composed of prostaglandins, thromboxanes, lipoxins, leukotrienes, as well as epoxy- and hydroxy-modified arachidonic acids (EETs and HETEs, respectively) and is involved in regulation of local blood flow (2Capdevila J.H. Falck J.R. Biochem. Biophys. Res. Commun. 2001; 285: 571-576Crossref PubMed Scopus (116) Google Scholar), activity of smooth muscle cells (3Fang X. Weintraub N.L. Stoll L.L. Spector A.A. Hypertension. 1999; 34: 1242-1246Crossref PubMed Scopus (58) Google Scholar, 4Fleming I. Circ. Res. 2001; 26: 753-762Crossref Scopus (321) Google Scholar), secretion of cytokines (5Planaguma A. Titos E. Lopez-Parra M. Gaya J. Pueyo G. Arroyo V. Claria J. FASEB J. 2002; 16: 1937-1939Crossref PubMed Scopus (58) Google Scholar), cell proliferation (6Nie D. Che M. Grignon D. Tang K. Honn K.V. Cancer Metastasis Rev. 2001; 20: 195-206Crossref PubMed Scopus (96) Google Scholar), and cell migration (7Glenn H.L. Jacobson B.S. Cell Motil. Cytoskeleton. 2003; 55: 265-277Crossref PubMed Scopus (24) Google Scholar) and aggregation (8Stockton R.A. Jacobson B.S. Mol. Biol. Cell. 2001; 12: 1937-1956Crossref PubMed Scopus (51) Google Scholar). The activities of AA derivatives are implicated in a number of physiological processes including inflammation, anaphylaxis, and hypertension (9Seeds M.C. Bass D.A. Clin. Rev. Allergy Immunol. 1999; 17: 5-26Crossref PubMed Scopus (61) Google Scholar, 10Parnes S.M. Expert Opin. Pharmacother. 2002; 3: 33-38Crossref PubMed Scopus (5) Google Scholar, 11Moreno C. Maier K.G. Hoagland K.M. Yu M. Roman R.J. Am. J. Hypertens. 2001; 14: 90S-97SCrossref PubMed Google Scholar). The identification and characterization of the enzymatic processes involved in generating and metabolizing these important signaling molecules are critical for understanding the role of these molecules in health and disease. Arachidonic acid is an abundant component of cell membrane phospholipids and is released by phospholipase A2 in response to extracellular signals (12Bazan N.G. Tu B. Rodriguez de Turco E.B. Prog. Brain Res. 2002; 135: 175-185Crossref PubMed Scopus (59) Google Scholar). All mammalian cells except erythrocytes convert AA into bioactive eicosanoids (13Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3070) Google Scholar) using some or all of the following three enzymatic pathways: 1) the cyclooxygenase pathway that leads to the synthesis of numerous prostaglandins, prostacyclins, and thromboxanes (14Imig J.D. Am. J. Physiol. 2000; 279: F965-F981Crossref PubMed Google Scholar, 15Ziboh V.A. Cho Y. Mani I. Xi S. Arch. Pharm. Res. 2002; 25: 747-758Crossref PubMed Scopus (48) Google Scholar); 2) the lipoxygenase pathway that results in accumulation of hydroperoxides (HPETEs) and leukotrienes (14Imig J.D. Am. J. Physiol. 2000; 279: F965-F981Crossref PubMed Google Scholar, 15Ziboh V.A. Cho Y. Mani I. Xi S. Arch. Pharm. Res. 2002; 25: 747-758Crossref PubMed Scopus (48) Google Scholar); 3) the AA monooxygenase pathway that creates EETs and HETEs and consists of P450s possessing epoxygenase, lipoxygenase-like, or ω/ω-1 hydroxylase activity (16Capdevila J.H. Harris R.C. Falck J.R. Cell Mol. Life Sci. 2002; 59: 780-789Crossref PubMed Scopus (65) Google Scholar). The first two enzymatic cascades involve multiple P450s responsible for the synthesis of secondary eicosanoids. Cytochrome P450 epoxygenases synthesize four regio-epoxy isomers 5, 6-EET, 8, 9-EET, 11,12-EET and 14,15-EET (16Capdevila J.H. Harris R.C. Falck J.R. Cell Mol. Life Sci. 2002; 59: 780-789Crossref PubMed Scopus (65) Google Scholar), which can be further converted by epoxide hydrolases to corresponding dihydroxyeicosatrienoic acids (17Fang X. Kaduce T.L. Weintraub N.L. Harmon S. Teesch L.M. Morisseau C. Thompson D.A. Hammock B.D. Spector A.A. J. Biol. Chem. 2001; 276: 14867-14874Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Lipoxygenase-like P450s create 5-HETE, 8-HETE, 9-HETE, 11-HETE, 12-HETE, or 15-HETE (16Capdevila J.H. Harris R.C. Falck J.R. Cell Mol. Life Sci. 2002; 59: 780-789Crossref PubMed Scopus (65) Google Scholar). ω/ω-1-Hydroxylase converts AA into 20-HETE, 19-HETE, 18-HETE, 17-HETE, or 16-HETE (18Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Crossref PubMed Scopus (139) Google Scholar). All of the above products can be modified further into additional eicosanoids. Arachidonic acid monooxygenase activity was first characterized in microsomal fractions obtained from kidney and liver (18Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Crossref PubMed Scopus (139) Google Scholar). More recently, numerous tissue-specific AA-metabolizing P450s have been identified in human and animal tissues. For example, human hepatic CYP1A2 (19Rifkind A.B. Lee C. Chang T.K. Waxman D.J. Arch. Biochem. Biophys. 1995; 320: 380-389Crossref PubMed Scopus (219) Google Scholar, 20Bylund J. Kunz T. Valmsen K. Oliw E.H. J. Pharmacol. Exp. Ther. 1998; 284: 51-60PubMed Google Scholar) creates 14,15-EET, 11,12-EET, 8,9-EET, 7-HETE, 10-HETE, 13-HETE, and 19-HETE, whereas hepatic 2C19 (20Bylund J. Kunz T. Valmsen K. Oliw E.H. J. Pharmacol. Exp. Ther. 1998; 284: 51-60PubMed Google Scholar, 21Bylund J. Ericsson J. Oliw E.H. Anal. Biochem. 1998; 265: 55-68Crossref PubMed Scopus (120) Google Scholar) synthesizes 8,9-EET, 14,15-EET, 19-HETE, and 20-HETE. Other P450s convert AA into a more discrete set of products. Human CYP2J2 activity results in the accumulation of all four EETs (22Wu S. Moomaw C.R. Tomer K.B. Falck J.R. Zeldin D.C. J. Biol. Chem. 1996; 271: 3460-3468Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar), rat CYP4A1 creates predominantly 20-HETE (23Aoyama T. Hardwick J.P. Imaoka S. Funae Y. Gelboin H.V. Gonzalez F.J. J. Lipid Res. 1990; 31: 1477-1482Abstract Full Text PDF PubMed Google Scholar) and murine CYP2J9, which is highly expressed in cerebellar Purkinje cells, synthesizes exclusively the bioactive AA metabolite 19-HETE (24Qu W. Bradbury J.A. Tsao C.C. Maronpot R. Harry G.J. Parker C.E. Davis L.S. Breyer M.D. Waalkes M.P. Falck J.R. Chen J. Rosenberg R.L. Zeldin D.C. J. Biol. Chem. 2001; 276: 25467-25479Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). An important physiological role has been demonstrated in the brain for 19-HETE, which can inhibit the activity of recombinant P/Q-type Ca2+ channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release (24Qu W. Bradbury J.A. Tsao C.C. Maronpot R. Harry G.J. Parker C.E. Davis L.S. Breyer M.D. Waalkes M.P. Falck J.R. Chen J. Rosenberg R.L. Zeldin D.C. J. Biol. Chem. 2001; 276: 25467-25479Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Tissue- and substrate-specific P450 monooxygenases have gained attention as essential control points in AA signal transduction pathways. In this paper, we report the cloning of a novel human P450, CYP2U1, which is expressed predominantly in thymus and cerebellum. We have determined that CYP2U1 metabolizes AA, docosahexaenoic acid (DHA), and other long chain fatty acids to a series of oxygenated products and might play a significant role in brain and immune functions. Materials—[α-32P]dATP was purchased from PerkinElmer Life Sciences. 20-Hydroxy-AA was purchased from Cayman Chemical (Ann Arbor). [1-14C]Arachidonic, docosahexaenoic, linolenic, eicosapentaenoic (EPA), docosatraenoic (DTA), and eicosatrienoic (ETA) acids and all other chemicals were purchased from Sigma unless specified. Full-length cDNA Cloning and Sequencing—Human EST, genomic and High Throughput Genomic Sequence data bases (NCBI, Bethesda, MD) were searched using TBLASTN, BLASTN, and BLASTX algorithms. EST clone AI216236 and genomic clone AC000016 were identified and purchased from Research Genetics (Birmingham, AL). Custom oligonucleotide synthesis and DNA sequencing were performed by Cortec (Kingston, ON, Canada). A human thymus 5′-STRETCH Plus cDNA library (Clontech) was screened according to the manufacturer's instructions. A [α-32P]dATP-radiolabeled probe was prepared by random priming using as template a purified 380-bp fragment of EST clone AI216236 corresponding to nucleotides 1400-1779 (accession no. AY343323). One clone was identified, and based on the sequence data this clone lacked the 5′- and 3′-ends. To obtain the full-length cDNA sequence of CYP2U1, the 5′- and 3′-ends were amplified using the SMART RACE cDNA amplification kit (as per the manufacturer's instructions, Clontech) and thymus cDNA as template. CYP2U1 specific and were and used the in the amplification PCR products were into a (Clontech) and The full-length clone of CYP2U1 (accession no. was from the using a kit as per the manufacturer's instructions. multiple of P450 the by and Google Scholar) was used a and a of and PCR of CYP2U1 in Human cDNA from human was purchased from Human thymus and cerebellum were obtained from and human kidney was obtained from of the were using random and according to the manufacturer's for human CYP2U1 were to specific amplification of a fragment of the The of these were as and real-time PCR was performed using PCR according to the manufacturer's PCR were in using cDNA was in PCR was performed on an sequence a was performed on all PCR products to that specific PCR products were The real-time PCR results were using the sequence The CYP2U1 expression were using the and to or expression of Recombinant CYP2U1 in cDNA of CYP2U1 and CYP2U1 were into Full-length human cytochrome and cytochrome P450-NADPH were from thymus by specific The and and the and were used for PCR amplification of and hOR, were into a Sf9 insect cells were of the expression using the expression according to manufacturer's a the DNA was Recombinant were and the of CYP2U1, and was by PCR analysis. Sf9 insect cells were in in were acid, and and respectively) to recombinant cells were and microsomal were prepared as by Zeldin D.C. Falck J.R. J.H. Arch. Biochem. Biophys. 1995; PubMed Scopus Google Scholar). protein was by the acid were in in liquid The of a functional CYP2U1 protein in the microsomal was by analysis. of CYP2U1 were corresponding to acids of the CYP2U1 synthesis and were by Research Genetics AL). Sf9 insect cells and Sf9 cells were and in were performed according to the manufacturer's and or of and cell microsomal of were in to was in The was for in a The were by the addition of and for were and acid. An of acid) was to and the were The metabolites were by were as above in the of or 17-octodecynoic acid and was performed using a mass to a a The was a and was using a a flow of The of and acid The were set and C. was to a flow of C. The was for and to the For of the following a flow of was were set and for was to a flow of The was for a of and C. was to in The was for to the and for The mass was in a and was used as a flow was a flow of flow of a of and a of For was used as a a of of V. The mass were and the was V. The and the mass were by of AA into the and of acid) The mass was in a mass and of AA were characterized by of the from Sf9 cells were as above and fatty acids. In these AA was for of EPA, DHA, DTA, ETA, and acid and on a to these AA for the Cloning of CYP2U1 an to novel human we searched the human EST data using a sequence for the P450 One of the identified (accession no. a genomic sequence (accession no. that was in a of the human genomic data To which tissue be used for the full-length cloning of this novel human P450, we screened a human blot from a of and cell using a probe from the EST clone A signal was on from and thymus tissue not we screened a human thymus cDNA library that in an of full-length cDNA acids The acid sequence of the of cytochrome P450s D. R. Biol. 2000; PubMed Google are the acids from to which are in the the in to the to which an Y. 1998; PubMed Scopus Google Scholar), using and from the and from the sequence was also found acids the The CYP2U1 for this was by the Cytochrome P450 CYP2U1 has 1) and from most of the family which have In the CYP2U1 the of 2, and is other of the the is The analysis of CYP2U1 acid sequence that has the of to AA and and S. Parker C. Zeldin D.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). A of acid sequence other human that CYP2U1 is most related to CYP2J2 D.C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), an AA and a microsomal D.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and acid sequence of CYP2U1 expression of CYP2U1 was determined in the human by real-time PCR These that CYP2U1 was most abundant in the thymus and cerebellum and in numerous other tissues. The analysis that CYP2U1 expression in thymus is in the cerebellum and about other tissues. Recombinant of CYP2U1 in Sf9 the and enzymatic activity of CYP2U1, we used to CYP2U1 A recombinant protein was and in Sf9 insect The protein expression was by blot and analysis. The a protein in the microsomal of Sf9 cells In the of CYP2U1 protein analysis using microsomal from Sf9 cells CYP2U1 a these we the of of recombinant CYP2U1 protein per of protein in microsomal of Arachidonic by CYP2U1 and family to the role of CYP2U1 in fatty acid fractions recombinant CYP2U1 protein were in the of AA and by Arachidonic acid was by CYP2U1 in a and an of the of two more metabolites of AA as in as and These were identified based on The mass of the two products is in The to a of oxygenated derivatives of AA The of the two oxygenated derivatives is in A and B. For the fragment and The fragment from the of and of The of fragment and which were by and J. Ericsson J. Oliw E.H. Anal. Biochem. 1998; 265: 55-68Crossref PubMed Scopus (120) Google Scholar) to be for metabolizes AA to 19- and 20-HETE. of the two oxygenated derivatives identified as and (as in 19- and 20-HETE The of the metabolite 2) fragment and two signals and J. Ericsson J. Oliw E.H. Anal. Biochem. 1998; 265: 55-68Crossref PubMed Scopus (120) Google Scholar). The fragment might be the of the of and The and of this metabolite 2) were the of 20-HETE. the of a the of as 20-HETE not these was that CYP2U1 converts AA into two bioactive derivatives, 19- and 20-HETE. CYP2U1 the of fatty acids to CYP2U1, using and to of fatty acids were In these we a in AA acid, EPA, DHA, and were to the their ability to AA for the of CYP2U1 not To further the of CYP2U1, of fatty acids chain or of were analysis demonstrated that acid, EPA, ETA, and are substrates of recombinant CYP2U1 and and In the of DHA, the of more metabolites by The two metabolites have a in of and for A and In to the mass to of the the for these two metabolites is to which to the addition of Based on this we postulate that the metabolites to a and or to an such as acid. of the of the identification of metabolites not be CYP2U1 not for lauric acid or linoleic acid the addition of high to of not of acid, EPA, and by from Sf9 cells or were acid or as the products were and by The mass was in a mass To the following were and and and and These to the and oxygenated substrates Sf9 cell microsomal CYP2U1 were in the or of for in a the metabolites were by The abbreviations are: not by by not of of acid acid acid acid acid acid acid in a new We also fatty acids in insect cell be by To this we performed analysis using recombinant CYP2U1 or analysis that CYP2U1 was to metabolize and or and docosahexaenoic long chain fatty acids to more products and For most of these fatty two metabolites were recombinant For of the metabolites the was the of a to the of a or acid. on the that is known to be a P450 and a specific P450 we the ability of these two to inhibit CYP2U1 We used of or in the of microsomal fractions from Sf9 cells, and the of AA of or 17-ODYA, for of and were This the enzymatic activities of CYP2U1, the first of this novel of cytochrome P450s to be This also that CYP2U1 plays critical roles in brain and thymus We have that CYP2U1 metabolizes AA to two bioactive metabolites 19- and 20-HETE and demonstrated that CYP2U1 was to metabolize or long chain fatty acids to metabolites that be involved in signaling pathways. the specific expression of this was in thymus as well as cerebellum. CYP2U1 protein and acid sequence two AA and S. Parker C. Zeldin D.C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the CYP2U1 is on whereas other family are on This might that has recently for this the of other family the CYP2U1 is well and acid sequence and rat We have that CYP2U1 can metabolize or long chain fatty acids The of chain fatty acid was also but results that lauric acid was not a of CYP2U1 the of this P450 for fatty acids A fatty acid chain has been for P450s in K. E. Y. K. S. Y. M. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), I. G. J.P. Biochem. Biophys. Res. Commun. 1998; PubMed Scopus Google Scholar) and J.A. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). the CYP2U1 long chain fatty acid to be in the of AA has been for of the and data that CYP2U1 has the ability to AA into two 19- and 20-HETE, characterized as bioactive In of the family have also been characterized to AA the and physiological in the regulation of have been for 19- and 20-HETE. In kidney and brain for example, 20-HETE has been characterized as a R.J. Maier K.G. M. Clin. Exp. Pharmacol. Physiol. 2000; PubMed Scopus Google Scholar, Zeldin D.C. Opin. 2002; PubMed Scopus Google Scholar). In rat and Maier K.G. S. D. Roman R.J. Am. J. Physiol. 2002; PubMed Scopus Google Scholar) have that 20-HETE to the in blood flow 20-HETE, as well as 19-HETE, has been to channels in smooth muscle B. Falck J.R. J. Pharmacol. 1990; 16: PubMed Scopus Google Scholar). 19-HETE can inhibit the activity of recombinant P/Q-type Ca2+ channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release B. Falck J.R. J. Pharmacol. 1990; 16: PubMed Scopus Google Scholar). A highly expressed P450 in murine cerebellar Purkinje cells, has been to metabolize exclusively AA to 19-HETE (24Qu W. Bradbury J.A. Tsao C.C. Maronpot R. Harry G.J. Parker C.E. Davis L.S. Breyer M.D. Waalkes M.P. Falck J.R. Chen J. Rosenberg R.L. Zeldin D.C. J. Biol. Chem. 2001; 276: 25467-25479Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). the of 19- and 20-HETE CYP2U1 also play a role in controlling in specific and be involved in the regulation of blood in these We have demonstrated that CYP2U1 can also on fatty acids such as or which are of and of disease. data have been on the identification of a human cytochrome P450 involved in the of or In a member of the was to the of A. E. K. M. Biophys. Scholar). is the most fatty acid in and and has been found in high in and and have been to be the fatty acids found in signaling has also been in brain S. M. W. J. T. Science. 2000; PubMed Scopus Google Scholar). rat brain was to high activity of M. A. 1995; PubMed Scopus Google Scholar). the expression of CYP2U1 in cerebellum and ability to generate 19- and 20-HETE, we can postulate that might play a role in the regulation of channels or in cells or be involved in the of cerebellar blood The that CYP2U1 metabolizes to and and that is expressed in the cerebellum that CYP2U1 is a for the of in the We have that CYP2U1 is highly expressed in the a physiological role in of immune eicosanoids such as are known as and of immune cell of cells of the immune an of long chain fatty acids such as and have been to proliferation of or J. 2000; PubMed Scopus Google Scholar) and to the of and the J. 2000; PubMed Scopus Google Scholar, 1994; Google Scholar). CYP2U1 may play a significant role in modulating signal transduction expression and immune cells AA, DHA, in thymus. We and A. for We for the of this

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