Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions-128 and -70 (H. K. Das, T. Leff, and J.L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence -128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.
Apolipoprotein B-100, produced primarily in the human liver, is the sole protein component of low-density lipoprotein and serves as a ligand for the LDL receptor. Two cis-acting positive elements between −128 and −70 control hepatic cell-specific expression of the human apoB gene (H. K. Das, T. Leff, and J. L. Breslow, J. Biol. Chem. 263: 11452-11458, 1988). In this study, two apoB cis-acting elements (+20 to +40; +43 to +53) have been identified using DNase I footprint analysis. Through in vitro mutagenesis and transient transfection experiments in Hep G2 and HeLa cells, the element (+20 to +40) was observed to have a negative effect on transcription of the apoB gene. The element (+43 to +53) was found to have a strong positive effect on apoB gene transcription in Hep G2 cells and mild positive effect in HeLa cells. Therefore these two cis-acting elements mediate hepatic-cell specific expression of the apolipoprotein gene by interacting with trans-acting protein factors.
Natural killer (NK) cells spontaneously detect and kill cancerous and virally infected cells through receptors that transduce either activating or inhibiting signals. The majority of well studied NK receptors are involved in inhibitory signaling. However, we have previously described an activating receptor, 2B4, expressed on all murine NK cells and a subset of T cells that mediate non‐major histocompatibility complex (MHC) restricted killing. Anti‐2B4 monoclonal antibodies directed against IL‐2‐activated NK cells enhanced their destruction of tumor cells. Recently, we determined binding of 2B4 to CD48 with a much higher affinity than CD2 to CD48. Here we describe the molecular characterization of a cDNA clone homologous to mouse 2B4, isolated from a human NK cell library. The cDNA clone contained an open reading frame encoding a polypeptide chain of 365 amino acid residues. The predicted protein sequence showed 70% similarity to murine 2B4. Additionally, it has 48, 45, and 43% similarity to human CD84, CDw150 (SLAM), and CD48, respectively. RNA blot analysis indicates the presence of 3 kb and 5 kb transcripts in T‐ and NK‐cell lines. A single transcript of 3 kb is identified in poly(A) + RNA from human spleen, peripheral blood leukocytes, and lymph node, whereas, the level of expression in bone marrow and fetal liver was indeterminate. Preliminary functional data suggests that NK‐cell interaction with target cells via 2B4 modulates human NK‐cell cytolytic activity.
Summary 2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8 + T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non‐major histocompatibility complex (MHC)‐restricted killing. Recently, 9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non‐MHC‐restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon‐γ (IFN‐γ) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase‐2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.
A dozen 24-sulfoximine analogues of the hormone 1alpha,25-dihydroxyvitamin D(3) were prepared, differing not only at the stereogenic sulfoximine stereocenter but also at the A-ring. Although these sulfoximines were not active transcriptionally and were only very weakly antiproliferative, some of them are powerful hydroxylase enzyme inhibitors. Specifically, 24-(S)-NH phenyl sulfoximine 3a is an extremely potent CYP24 inhibitor (IC(50) = 7.4 nM) having low calcemic activity. In addition, this compound shows high selectivity toward the CYP24 enzyme in comparison to CYP27A1 (IC(50) > 1000 nM) and CYP27B (IC(50) = 554 nM).
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 primers 5′-GTT GCT TGG ATT CAC TGT CCT T-3′, 5′-GGT TTC TCC CAA ATG GCT GGG TCT CT-3′, and 5′-CCA CAG TTA GCC TCT GCA CTT CC-3′ were synthesized and used with the primers provided in the amplification kit. Unique PCR products were then cloned into a pTAdv vector (Clontech) and sequenced. The full-length clone of CYP2U1 (accession no. AY343323) was assembled from the various fragments using a homologous recombination kit (Pangene, Fremont CA) as per the manufacturer's instructions. Phylogenetic Analyses—For multiple alignment of P450 sequences, the ClustalV method described by Higgins and Sharp (25Higgins D.G. Sharp P.M. Comput. Appl. Biosci. 1989; 5: 151-153PubMed Google Scholar) was used with a gap penalty and a gap length penalty of 10 and PAM250 residue weight table. Real-time PCR Analysis of CYP2U1 Expression in Human Tissues—A cDNA panel from 24 different human tissues was purchased from Origene Technologies Inc. (Rockville, MD). Human thymus and cerebellum poly(A) RNA samples were obtained from Clontech, and human kidney poly(A)+ RNA was obtained from Ambion (Austin, TX). Aliquots of the RNAs were reverse transcribed using random hexamers and Thermoscript reverse transcriptase according to the manufacturer's instructions (Invitrogen). Primers for human CYP2U1 were designed to ensure specific amplification of a 194-bp fragment of the cDNA. The sequences of these primers were as follows: forward, 5′-GCA TCG AGC AGA ACA TTC GCG CCA-3′; and reverse, 5′-CTG GTG CCT GCT GTA TAT GCT GCT G-3′. Quantitative real-time PCR was performed using SYBR Green PCR Master Mix according to the manufacturer's instructions (Qiagen, Valencia, CA) with minor revisions. Briefly, PCR amplifications were conducted in 25-μl reactions using 60 cycles. Each cDNA sample was tested in triplicate. Real-time PCR was performed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). After amplification, a dissociation curve was performed on all PCR products to ensure that specific PCR products were generated. The real-time PCR results were analyzed using the sequence detection system software V1.0 (Applied Biosystems). The CYP2U1 expression levels were calculated using the comparative CT method and normalized to β-actin (cDNA panel) or glyceraldehyde-3-phosphate dehydrogenase (mRNA) expression levels. Heterologous Expression of Recombinant CYP2U1 in Insect Cells— PCR-generated cDNA coding region of CYP2U1 (pVL-CYP2U1) and His6-tagged CYP2U1 (pVL-CYP2U1-His6) were subcloned into pVL1392 baculovirus transfer vector (BD Biosciences). Full-length human cytochrome b5 (hb5) and cytochrome P450-NADPH reductase (hOR) cDNAs were isolated from thymus total RNA by reverse transcription-PCR with specific primers. The forward primers, 5′-ATC CCG GGA TGG CAG AGC AGT CGG ACG AG-3′ and 5′-ATA GAT CTG AAA TGG GAG ACT CCC TGG AC-3′, and the reverse primers, 5′-ATG GAT CCT CAG TCC TCT GCC ATG TAT AGG-3′ and 5′-ATG AAT TCC TAG CTC CAC ACG TCC AGG G-3′, were used for PCR amplification of hb5 and hOR, respectively. Both cDNAs were then subcloned together into a pAcDB3 transfer vector (BD Biosciences). Cultured Sf9 insect cells were cotransfected with each of the expression vectors using the Baculo-Gold baculovirus expression system according to manufacturer's instructions (BD Biosciences). As a control, the linearized wild-type Baculo-Gold viral DNA was used. Recombinant viruses were purified, and the presence of CYP2U1, hb5 cDNA, and hOR cDNAs was confirmed by PCR analysis. Cultured Sf9 insect cells were grown in Spinner flasks in TNM-FH medium. Cultures were supplemented with hemin, δ-aminolevulinic acid, and ferric citrate (final concentration 2 μg/ml, 100 μm, and 100 μm, respectively) just prior to infection with recombinant baculovirus. Seventy-two hours later, transfected cells were harvested, and crude microsomal preparations were prepared as described by Zeldin et al. (26Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar). Total protein concentration was estimated by the bicinchoninic acid method. Microsomal preparations were stored in small aliquots in liquid nitrogen. The presence of a functional CYP2U1 protein in the microsomal preparation was confirmed by reduced carbon monoxide difference spectroscopic analysis. Immunoblot Analysis of CYP2U1 Expression—Polyclonal rabbit antibodies were raised against keyhole limpet hemocyanin-conjugated oligopeptide IKDHQESLDRENPQD corresponding to amino acids 302-316 of the CYP2U1 protein. Peptide synthesis and immunizations were conducted by Research Genetics (Huntsville, AL). Sf9 insect cells and CYP2U1-transfected Sf9 cells were lysed and analyzed in 10% SDS-polyacrylamide gels (under reducing conditions). Western blots were performed according to the manufacturer's chemiluminescence detection system instructions (Amersham Biosciences) and hybridized with rabbit anti-CYP2U1 polyclonal antibody or anti-His6 monoclonal antibody (Invitrogen). Metabolism of Fatty Acids and Product Characterization—Sf9 cell microsomal preparations (0.1-1 mg of protein/ml final concentration) were diluted in assay buffer (0.1 m potassium phosphate, pH 7.4, 5 mm MgCl2, 1 mm EDTA). When necessary, exogenous substrate (up to 100 μm final concentration) was added in ethanol solution. The mixture was preincubated at 37 °C for 5 min in a water bath under constant mixing. The reactions were started by the addition of NADPH-regenerating mix (1 mm NADPH, 1 unit/ml glucose-6-phosphate dehydrogenase, 2 mm glucose 6-phosphate, final concentration) and carried out for 5-60 min at 37 °C. Aliquots were taken and stopped with acetic acid. An internal standard (100 ng of myristic acid) was added to each reaction, and the samples were extracted twice with acidified ethyl acetate. The metabolites were analyzed by LC-MS. Inhibition studies were conducted as described above in the presence of 0.1-50 μm ketoconazole or 17-octodecynoic acid (17-ODYA). HPLC and MS Analysis—LC-MS was performed using a Micromass Quattro Ultima triple-stage quadrupole mass spectrometer (Manchester, UK) connected to a Waters Alliance 2969 HPLC system equipped with a photodiode array UV (200-400 nm) detector (Milford, MA). The analytical column was a Zorbax C18 Eclipse XDB 150 × 2.1 mm (inner diameter) (5-μm particle size) (Agilent Technologies, Palo Alto, CA) and maintained at 25 °C. Fast separation was achieved using a 30-min HPLC linear gradient at a flow rate of 0.2 ml/min. The mobile phase consisted of water (solvent A), acetonitrile (solvent B), and 10% glacial acetic acid (solvent C). The initial mobile phase conditions were set at 39.5% A, 59.5% B, and 1% C. Solvent B was then increased to 99% with a constant flow of 1% C. The mobile phase was then held isocratically for 10 min at 99% B and 1% C before returning to the initial conditions. For better separation of metabolites, the following HPLC linear gradient at a flow rate of 0.2 ml/min was used. Initial mobile phase conditions were set at 54% A, 45% B, and 1% C for 1 min. Solvent B was increased to 55% B with a constant flow of 1% C at 30 min. The mobile phase was then held isocratically for 10 min at a composition of 44% A, 55% B, and 1% C. Solvent B was increased to 99% in 5 min while maintaining C at 1%. The mobile phase was held isocratically for 15 min before returning to the initial conditions over 5 min and equilibrating for 5 min. The mass spectrometer was operated in negative electrospray mode with a -2.86-kV capillary voltage and -16-V cone voltage. Nitrogen was used as a drying gas. Solvent flow rate was fixed at 200 μl/min with a cone gas flow of 112 liters/h, desolvation gas flow of 778 liters/h, a source temperature of 149 °C, and a desolvation temperature of 200 °C. For tandem MS experiments, argon was used as a collision gas at a pressure of 2.3 × 10-3 torr with collision energy of 22 V. The quadrupole mass resolution settings were fixed at 15.0, and the multiplier voltage was 650 V. The ion source and the mass spectrometer parameters were optimized by infusion of 10 μl/min AA standard solution into the HPLC eluent (75% acetonitrile, 24% water, and 1% of 10% glacial acetic acid) flowing at 200 μl/min. The mass spectrometer was operated in a full mass scan (m/z 150-500) and product ion scan mode. Metabolites of AA were characterized by performing product ion scans of the molecular ion. Competition Assay—Microsomes isolated from CYP2U1-transfected Sf9 cells were incubated as described above with 2.5 μm [1-14C]AA and 0-25 μm unlabeled fatty acids. In these experiments, AA metabolism was measured for each concentration of EPA, DHA, DTA, ETA, and linolenic acid and plotted on a graph to determine whether these compounds compete with AA for the substrate binding site. Molecular Cloning of CYP2U1 cDNA—In an effort to discover novel human P450s, we searched the human EST data base using TBLASTN with a consensus sequence for the P450 heme binding domain PF(G/S)XGX(A/R/H)XCXG. One of the identified sequences (accession no. AI216236) closely matched a partial genomic sequence (accession no. AC00016) that was selected in a similar search of the human genomic data base. To identify which tissue RNA should be used for the full-length cloning of this novel human P450, we screened a human RNA dot blot containing poly(A)+ RNA samples from a variety of tissues and cell lines using a 381-bp probe from the EST clone AI216236. A strong hybridization signal was detected on dot blots loaded with mRNA from adult and fetal thymus tissue (data not shown). Subsequently, we screened a human thymus cDNA library that resulted in an assembly of full-length cDNA (3,544 nucleotides) encoding 544 amino acids (Fig. 1). The predicted amino acid sequence contains many of the hallmark features of cytochrome P450s (27Werck-Reichhart D. Feyereisen R. Genome. Biol. 2000; 1 (REVIEWS3003)Crossref PubMed Google Scholar): heme binding domain PFGIGKRVCMGA (underlined are the amino acids from position 482 to 492 which are conserved in the heme binding domain (with the axial Cys ligand in bold to the heme)); PERF domain (PNRF position 464 to 467) which forms an ERR triad (28Zheng Y.M. Fisher M.B. Yokotani N. Fujii-Kuriyama Y. Rettie A.E. Biochemistry. 1998; 37: 12847-12851Crossref PubMed Scopus (55) Google Scholar), using Glu and Arg from the K-helix domain and Arg from the PERF domain; ETLR domain (EVQR) conserved sequence was also found 50 amino acids upstream the PERF domain. The CYP2U1 designation for this gene was approved by the Cytochrome P450 Nomenclature Committee (Dr. David Nelson Web Page: www.drnelson.utmem.edu/CytochromeP450.html). CYP2U1 gene has five exons (Fig. 1) and therefore differs from most of the CYP2 family members, which have nine exons. In the CYP2U1 gene, the position of introns 1, 2, and 4 is conserved with other members of the CYP2 family; however, the third intron position is different. The alignment analysis of CYP2U1 amino acid sequence reveals that it has the highest percentages of identity to fish AA epoxygenases, CYP2N1 and CYP2N2, with 39.6 and 38.4% identity, respectively (29Oleksiak M.F. Wu S. Parker C. Karchner S.I. Stegeman J.J. Zeldin D.C. J. Biol. Chem. 2000; 275: 2312-2321Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A phylogenetic comparison of amino acid sequence with other human CYPs showed that CYP2U1 is most closely related to CYP2J2 (30Zeldin D.C. J. Biol. Chem. 2001; 276: 36059-36062Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), an AA epoxygenase gene, and CYP2R1, a microsomal vitamin D 25-hydroxylase (31Cheng J.B. Motola D.L. Mangelsdorf D.J. Russell D.W. J. Biol. Chem. 2003; 278: 38084-38093Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) with 36 and 37.1% amino acid sequence identity, respectively (Fig. 2). Tissue Distribution of CYP2U1 mRNA—The expression of CYP2U1 mRNA was determined in the human by real-time PCR (Fig. 3). These experiments revealed that CYP2U1 mRNA was most abundant in the thymus and cerebellum and present at lower levels in numerous other tissues. The quantitative analysis showed that CYP2U1 expression in thymus is ∼3-fold higher than in the cerebellum and about 200 magnitudes higher compared with other tissues. Recombinant Expression of CYP2U1 in Sf9 Insect Cells—To characterize the substrate and enzymatic activity of CYP2U1, we used baculovirus to express CYP2U1 protein. A recombinant CYP2U1-His6 protein was coexpressed with hOR and hb5 in Sf9 insect cells. The protein expression was verified by Western blot and spectral analysis. The anti-His6 antibody cross-reacted with a ∼55-60-kDa protein present in the microsomal fraction of infected Sf9 cells (Fig. 4A). In addition, anti-CYP2U1 antibody confirmed the presence of CYP2U1 protein (Fig. 4B). Spectral analysis using microsomal fraction from Sf9 cells infected with CYP2U1 baculovirus showed a characteristic carbon monoxide/reduced 450 nm absorption peak (Fig. 4C). From these data, we estimated the presence of 0.5 nmol of recombinant CYP2U1 protein per mg of total protein in microsomal fractions. Metabolism of Arachidonic Acid by CYP2U1—Sequence similarities between CYP2U1 and others CYP2 family members prompted us to study the potential role of CYP2U1 in fatty acid metabolism. Microsomal fractions containing recombinant CYP2U1 protein were incubated in the presence of 10 μm exogenous AA and analyzed by LC-MS at different time intervals. Arachidonic acid was metabolized by CYP2U1 in a time-dependent manner and with an apparent Km value of 2.7 μm. LC-MS chromatograms revealed the presence of two more polar, water-soluble metabolites of AA as indicated in Fig. 5 as peak 1 and peak 2. These compounds were identified based on MS data. The negative electrospray mode mass spectrum of the two products is shown in Fig. 6. The [M-H]- ion at m/z 319 corresponds to a single carboxylate anion of oxygenated derivatives of AA (HETE). The product ion spectrum of the two oxygenated derivatives is shown in Fig. 6, A and B. For peak 1, the product ion spectrum yielded fragment ions at m/z 301, 275, 259, 231, 203, 177, 135, 121, and 107. The fragment at m/z 231 probably arises from the combined loss of CO2 and CH2OH (see inset of Fig. 6A). The product ion spectrum of peak 1 yields characteristic fragment ions at m/z 275 and 231, which were reported previously by Bylund and coauthors (21Bylund J. Ericsson J. Oliw E.H. Anal. Biochem. 1998; 265: 55-68Crossref PubMed Scopus (120) Google Scholar) to be characteristic fragments for 19-HETE.Fig. 6CYP2U1 metabolizes AA to 19- and 20-HETE. Product ion spectrum of the two oxygenated derivatives identified as peak 1 (A) and peak 2 (B) (as shown in Fig. 5). Drawings represent predicted 19- and 20-HETE fragmentation scenarios, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The product ion spectrum of the second metabolite (peak 2) yielded fragment ions at m/z 301, 289, 275, 257, 245, 109, 107, and 97, with two characteristic signals at m/z 289 and 245 (21Bylund J. Ericsson J. Oliw E.H. Anal. Biochem. 1998; 265: 55-68Crossref PubMed Scopus (120) Google Scholar). The fragment at m/z 245 might be the result of the combined loss of CO2 and CH2O (see inset, Fig. 6B). The retention time and product ion spectrum of this metabolite (peak 2) were consistent with the formation of 20-HETE. Moreover, the use of a reference confirmed the identity of peak 2 as 20-HETE (data not shown). From these observations, it was confirmed that CYP2U1 converts AA into two bioactive derivatives, 19- and 20-HETE. CYP2U1 Substrate Specificity—To assess the substrate binding properties of different fatty acids to CYP2U1, competition experiments using 2.5 μm [1-14C]AA and different concentrations (up to 25 μm) of unlabeled fatty acids were conducted. In these experiments, we measured a concentration-dependent decrease in AA metabolism when linolenic acid, EPA, DHA, and ETA were added to the incubation, confirming their ability to compete with AA for the binding site of CYP2U1 (data not shown). To characterize further the substrate specificity of CYP2U1, incubations of fatty acids with different chain lengths or degrees of saturation were conducted. HPLC analysis demonstrated that linolenic acid, EPA, ETA, and DHA are substrates of recombinant CYP2U1 (Figs. 7 and 8 and Table I). In the case of DHA, Fig. 7 shows the LC-MS chromatogram of more polar metabolites generated by CYP2U1. The two metabolites have a retention time in our HPLC conditions of 26.9 and 37 min, for peak A and B, respectively. In contrast to the mass to charge ratio of the deprotonated DHA ([M-H]- = 327), the [M-H]- for these two metabolites is equal to 343, which could correspond to the addition of one molecular oxygen ([M-H]- + 16). Based on this observation, we postulate that the metabolites formed correspond to a ω- and (ω-1)-hydroxylated DHA or to an epoxy-DHA such as 19,20-epoxydocosapentaenoic acid. Because of the absence of available reference compounds, the identification of metabolites formed could not be established. Moreover, CYP2U1 did not show an
In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.
