Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Δ6-desaturase activity
2004; Elsevier BV; Volume: 45; Issue: 1 Linguagem: Inglês
10.1194/jlr.m300339-jlr200
ISSN1539-7262
AutoresHervé Guillou, Sabine d’Andrea, Vincent Rioux, Romain Barnouin, Stéphanie Dalaine, Frédérique Pédrono, Sophie Jan, Philippe Legrand,
Tópico(s)Pancreatic function and diabetes
ResumoThe Δ6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Δ6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Δ6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Δ6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif.These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Δ6-desaturase activity. The Δ6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Δ6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Δ6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Δ6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Δ6-desaturase activity. Long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3) play pivotal roles in a variety of biological functions (1Spector A.A. Essentiality of fatty acids.Lipids. 1999; 34: 1-3Crossref PubMed Google Scholar). In animals, some of the daily needs in long-chain PUFAs are fulfilled from the diet. However, most of the long-chain PUFAs found in animal tissues are derived from the biosynthetic pathway involving elongations, Δ6-desaturation, and Δ5-desaturation for conversion of essential fatty acid precursors (C18:2n-6 and C18:3n-3) to their respective 20- and 22-carbon polyenoic products. None of the desaturases involved in this biosynthetic pathway have been reproducibly purified, and their structure remains to be characterized. The only animal desaturase whose structure is known is the Δ9-desaturase (2Heinemann F.S. Ozols J. Stearoyl-CoA desaturase, a short-lived protein of endoplasmic reticulum with multiple control mechanisms.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 123-133Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This enzyme is part of a multienzyme system present in the endoplasmic reticulum and is composed of Δ9-desaturase, NADH cytochrome b5 reductase, and cytochrome b5. In the process of double bond formation, the membrane-bound cytochrome b5 transfers electrons by lateral diffusion from NADH cytochrome b5 reductase to the Δ9 fatty acid desaturase (2Heinemann F.S. Ozols J. Stearoyl-CoA desaturase, a short-lived protein of endoplasmic reticulum with multiple control mechanisms.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 123-133Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Although the first mammalian Δ9-desaturase was cloned almost 20 years ago (3Thiede M.A. Ozols J. Strittmatter P. Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase.J. Biol. Chem. 1986; 28: 13230-13235Abstract Full Text PDF Google Scholar), mammalian desaturases involved in PUFA biosynthetic pathways, i.e., Δ6- and Δ5-desaturases, have been cloned more recently (4Cho H.P. Nakamura M.T. Clarke S.D. Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase.J. Biol. Chem. 1999; 274: 471-477Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 5Aki T. Shimada Y. Inagaki K. Higashimoto H. Kawamoto S. Shigeta S. Ono K. Suzuki O. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase.Biochem. Biophys. Res. Commun. 1999; 255: 575-579Crossref PubMed Scopus (111) Google Scholar, 6Cho H.P. Nakamura M.T. Clarke S.D. Cloning, expression, and fatty acid regulation of the human delta-5 desaturase.J. Biol. Chem. 1999; 274: 37335-37339Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 7Zolfaghari R. Cifelli C.J. Banta M.D. Ross A.C. Fatty acid delta(5)-desaturase mRNA is regulated by dietary vitamin A and exogenous retinoic acid in liver of adult rats.Arch. Biochem. Biophys. 2001; 391: 8-15Crossref PubMed Scopus (0) Google Scholar, 8Matsuzaka T. Shimano H. Yahagi N. Amenyia-Kudo M. Yoshikawa T. Hasty A.H. Tamura Y. Osuga J. Okazaki H. Lizuka Y. Takahashi A. Sone H. Gotoda T. Ishibashi S. Yamada N. Dual regulation of mouse delta-5-desaturase and delta (6)-desaturase gene expression by SREBP-1 and PPAR.J. Lipid Res. 2002; 43: 107-114Abstract Full Text Full Text PDF PubMed Google Scholar). Comparison of their respective amino acid sequences shows one major difference between Δ9-desaturase and Δ6- and Δ5-desaturases: an N-terminal cytochrome b5-like domain is present in Δ6- and Δ5-desaturases but not in Δ9-desaturase. Numerous cytochrome b5-like domains have been identified in various desaturases from yeast, plants, and animals (9Napier J.A. Michaelson L.V. Sayanova O. The role of cytochrome b5 fusion desaturases in the synthesis of polyunsaturated fatty acids.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 135-143Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This remarkable characteristic raises the possibility that NADH cytochrome b5 reductase transfers electrons to the catalytic site of these cytochrome b5 fusion desaturases directly via the cytochrome b5-like domain and does not require an independent cytochrome b5. The presence of such cytochrome b5-like domains in desaturase proteins is likely to have originated from a fusion with an ancestral cytochrome b5 gene that may have conferred some evolutionarily selectable advantage. Although these cytochrome b5 fusion domains have diverged significantly, a typical HPGG motif has been conserved. This particular sequence forms an accessible heme binding core of the cytochrome b5-like domain (10Lederer F. The cytochrome b5-fold: an adaptable module.Biochimie. 1994; 76: 674-692Crossref PubMed Scopus (87) Google Scholar). Among desaturases fused to a cytochrome b5-like domain, the cytochrome b5 domain has been demonstrated to be essential for borage Δ6-desaturase (11Sayanova O. Shewry P. Napier J. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.Plant Physiol. 1999; 121: 641-646Crossref PubMed Scopus (63) Google Scholar) and a yeast Δ9-acyl-CoA-desaturase (12Mitchell A.G. Martin C.E. A novel cytochrome b5-like domain is linked to the carboxyl terminus of the Saccharomyces cerevisiae delta-9 fatty acid desaturase.J. Biol. Chem. 1995; 50: 29766-29772Abstract Full Text Full Text PDF Scopus (131) Google Scholar). The mammalian Δ6-desaturase, also named FADS2, has been cloned (4Cho H.P. Nakamura M.T. Clarke S.D. Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase.J. Biol. Chem. 1999; 274: 471-477Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 5Aki T. Shimada Y. Inagaki K. Higashimoto H. Kawamoto S. Shigeta S. Ono K. Suzuki O. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase.Biochem. Biophys. Res. Commun. 1999; 255: 575-579Crossref PubMed Scopus (111) Google Scholar), and its activity has been described (4Cho H.P. Nakamura M.T. Clarke S.D. Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase.J. Biol. Chem. 1999; 274: 471-477Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 5Aki T. Shimada Y. Inagaki K. Higashimoto H. Kawamoto S. Shigeta S. Ono K. Suzuki O. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase.Biochem. Biophys. Res. Commun. 1999; 255: 575-579Crossref PubMed Scopus (111) Google Scholar, 13D'Andrea S. Guillou H. Jan S. Catheline D. Thibault J-N. Bouriel M. Rioux V. Legrand P. The same rat delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis.Biochem. J. 2002; 364: 49-55Crossref PubMed Scopus (96) Google Scholar, 14de Antueno R.J. Knickle L.C. Smith H. Elliot M.L. Allen S.J. Nwaka S. Winther M.D. Activity of human delta5 and delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.FEBS Lett. 2001; 509: 77-80Crossref PubMed Scopus (99) Google Scholar, 15Guillou H. Rioux V. Catheline D. Thibault J-N. Bouriel M. Jan S. D'Andrea S. Legrand P. Conversion of hexadecanoic acid to hexadecenoic acid by rat delta6-desaturase.J. Lipid Res. 2003; 44: 450-454Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16Ge L. Gordon J.S. Hsuan C. Stenn K. Prouty S.M. Identification of the delta-6 desaturase of human sebaceous glands: expression and enzyme activity.J. Invest. Dermatol. 2003; 120: 707-714Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It was shown that expression of rat Δ6-desaturase (17Michinaka Y. Aki T. Inagaki K. Higashimoto H. Shimada Y. Nakajima T. Shimauchi T. Ono K. Suzuki O. Production of polyunsaturated fatty acids by genetic engineering of yeast.J. Oleo Sci. 2001; 5: 359-365Crossref Scopus (5) Google Scholar) in a yeast strain deficient for cytochrome b5 was sufficient to confer to host cells the capacity to convert C18:2n-6 to C18:3n-6. However, coexpression of cytochrome b5 increased the level of Δ6-desaturated fatty acid (17Michinaka Y. Aki T. Inagaki K. Higashimoto H. Shimada Y. Nakajima T. Shimauchi T. Ono K. Suzuki O. Production of polyunsaturated fatty acids by genetic engineering of yeast.J. Oleo Sci. 2001; 5: 359-365Crossref Scopus (5) Google Scholar) accumulation, suggesting that "free" cytochrome b5 is not strictly required but may play a role in Δ6-desaturation in this yeast model. In this study, we compared, in COS-7 cells, the activity of recombinant wild-type rat Δ6-desaturase with the activity of mutated recombinant enzymes in which the typical cytochrome b5 53HPGG56 motif has been mutated or deleted. We also investigated in COS-7 cells the role of coexpressed microsomal cytochrome b5 in Δ6-desaturase activity and its putative capacity to compensate for the essentialness of the 53HPGG56 motif in the Δ6-desaturase function reported here. cis-7,10,13,16,19-Docosapentaenoic acid (C22:5n-3) was purchased from Matreya (Pleasant Gap, PA). The characterized fatty acid methyl ester of C24:6n-3 (18Ishiara K. Murata M. Kaneniwa M. Saito H. Shinohara K. Maeda-Yamamoto M. Kawasaki K. Ooizumi T. Effect of tetracosahexaenoic acid on the content and release of histamine, and eicosanoid production in MC/9 mouse mast cell.Lipids. 1998; 33: 1107-1114Crossref PubMed Scopus (34) Google Scholar) was a generous gift from Dr. K. Ishihara (National Research Institute of Fisheries Science, Yokohama, Japan). Other unlabeled fatty acids were from Sigma (St. Quentin Fallavier, France). Radiolabeled [1-14C]18:3n-3 and [1-14C]18:0 (52 mCi/mmol) were purchased from Perkin Elmer Life Science (Paris, France). Fetal calf serum (FCS) was purchased from Perbio (Bezons, France). Solvents (HPLC grade) were purchased from Fisher Scientific (Elancourt, France). Other reagents were from Sigma. The anti-cytochrome b5 (rabbit) polyclonal antibody and the anti-myc (mouse) monoclonal antibody used in this study were generous gifts from Dr. N. Borgese (University of Milan, Italy) and Dr. S. Suire (The Babraham Institute, Cambridge, UK), respectively. The pCMV-HAHA expression vector (19Wotton D. Lo R.S. Lee S. Massagué J. A Smad transcriptional corepressor.Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar) was a generous gift from Dr. A. Atfi (Institut National de la Santé et de la Recherche Médicale U482, Hôpital Saint-Antoine, Paris, France). The pCMV/β-Gal expression vector was a generous gift from Dr. C. Diot (Unité Mixte de Recherche Institut National de la Recherche Agronomique-Ecole Nationale Supérieure Agronomique de Génétique Animale, Rennes, France). The plasmids constructed for the expression of rat Δ6-desaturase (referred to as pCMV/Δ6) have been previously described (13D'Andrea S. Guillou H. Jan S. Catheline D. Thibault J-N. Bouriel M. Rioux V. Legrand P. The same rat delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis.Biochem. J. 2002; 364: 49-55Crossref PubMed Scopus (96) Google Scholar). A plasmid coding for rat cytochrome b5 was constructed for expression in mammalian cells and is referred to as pcDNA3/cytb5. From the published (20Mitoma J. Ito A. The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum.EMBO J. 1992; 11: 4197-4203Crossref PubMed Scopus (102) Google Scholar) rat cytochrome b5 sequence (GenBank accession number D13205), oligonucleotide primers were designed to PCR amplify the entire coding sequence, with its stop codon using the high-fidelity Pfu polymerase from Promega (Lyon, France). The forward primer (5′-CAATGGATCCATGCCGGCCCACATGC-3′) included the translation start codon (boldface) and the BamHI restriction site (underlined). The reverse primer (5′-CGTGCTCGAGCTCAGCTACTCTTGTGGCT-3′) contained the translation stop codon (boldface) and the XhoI restriction site (underlined). The PCR product amplified from rat liver cDNA was treated with BamHI and XhoI before cloning into pcDNA3 (Invitrogen, San Diego, CA). The HindIII-SalI fragment containing the full-length rat cytochrome b5 cDNA was subcloned in frame from pcDNA3 into pCMV-HAHA. This construction is referred to as pCMV-HAHA/cytb5 and allows the expression of a cytochrome b5 fused N-terminally to a double hemagglutinin (HA) epitope. A plasmid coding for a C-terminally myc-tagged Δ6-desaturase was constructed using pCMV for expression in mammalian cells and is referred to as pCMV/Δ6myc. From the published rat Δ6-desaturase sequence (5Aki T. Shimada Y. Inagaki K. Higashimoto H. Kawamoto S. Shigeta S. Ono K. Suzuki O. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase.Biochem. Biophys. Res. Commun. 1999; 255: 575-579Crossref PubMed Scopus (111) Google Scholar) (GenBank accession number AB021980), oligonucleotide primers were designed to amplify, by PCR, the entire coding sequence with a deleted stop codon. The forward primer (5′-CAGTGGATCCATGGGGAAGGGAGGTA-3′) included the translation start codon (italics) and the NcoI restriction site (underlined). The reverse primer (5′-TGTGCGGCCGCTTTGTGGAGGTAGGCATCC-3′) corresponded to the C-terminal sequence of the protein (italics) without its stop codon and a NotI site (underlined). The PCR product amplified from rat liver cDNA was treated with NcoI and NotI before cloning into pCMV/myc/cyto (Invitrogen). Mutagenesis of the 53HPGG56 motif in the N-terminal cytochrome b5 domain of rat Δ6-desaturase was performed using a site-directed mutagenesis kit (QuickChange; Stratagene, Amsterdam, The Netherlands) according to the manufacturer's protocol. Three sets of two mutagenic primers were designed (Table 1). In each set, both primers are complementary to the opposite strands of pCMV/Δ6 and insert the desired mutation or deletion. These primers were used to delete the 53HPGG56 domain, to delete H53, or to replace H53 with an alanine, providing new expression vectors named pCMV/Δ6−53HPGG56, pCMV/Δ6−H53, or pCMV/Δ6−H53A, respectively.TABLE 1Primers used for site-directed mutagenesisMutagenic Primers (5′–3′)Mutation or DeletionName of the PlasmidCCAAATGGTCCCAGCGGGCCCCAGGGGGGCACCGTG and CACGGTGCCCCCCTGGGGCCCGCTGGGACCATTTGGAlanine substituted for H53pCMV/Δ6−H53ACCAAATGGTCCCAGCGGXCCAGGGGGGCACCGTG and CACGGTGCCCCCCTGGCCXGCTGGGACCATTTGGH53 deletedpCMV/Δ6−H53CCAAATGGTCCCAGCGGXXXXCACCGTGTCATCGGACAC and GTGTCCGATGACACGGTGXXXXCCGCTGGGACCATTTGG53HPGG56 deletedpCMV/Δ6−53HPGG56The mutagenic codon is indicated in boldface. The codon deletions are indicated (X). Open table in a new tab The mutagenic codon is indicated in boldface. The codon deletions are indicated (X). Sequences coding for Δ6-desaturase and Δ6-desaturase with deletion of the sequence corresponding to the 53HPGG56 motif were used for PCR amplification with oligonucleotide primers before subcloning into p3×Flag (Sigma). The forward primer (5′-GACTGAAGCTTATGGGGAAGGGAGGTA-3′) included the translation start codon (italics) and a HindIII restriction site (underlined). The reverse primer (5′-CATGCGGATCCTCATTTGTGGAGGTAGGCATCC-3′) contained the translation stop codon (italics) and a BamHI site (underlined). The PCR products were treated with HindIII and BamHI before cloning into p3×Flag. The plasmids are referred to as p3×Flag/Δ6 and p3×Flag/Δ6-53HPGG56 and allow the expression of N-terminally Flag-tagged desaturases. The integrity of the constructs and the presence of the desired deletions or mutations were assessed by DNA sequencing. COS-7 cells were routinely maintained at ∼50% confluence and were cultured in DMEM containing 10% FCS, 50 IU/ml penicillin, and 50 μg/ml streptomycin. The cells were split 1 day before transfection to 30% confluence and transfected the next day using the Easyject Plus electroporator (Equibio, Monchelsea, UK) according to the manufacturer's instructions. Briefly, 106 COS-7 cells in 0.8 ml of DMEM were mixed with 30 μg of purified plasmid, electroporated at 250 V and 1,500 μF with unlimited resistance, and seeded on a 10 cm dish containing culture medium. Coverslips containing the paraformaldehyde-fixed cells transfected with pCMV/Δ6myc were washed in PBS (150 mM NaCl and 5 mM Na phosphate, pH 7.4) and preincubated for 10 min on a drop of blocking buffer containing Triton X-100 (PBS containing 0.5% BSA and 0.1% Triton X-100). The cells were extensively washed with blocking buffer and incubated for 30 min with the primary antibody (monoclonal anti-Myc) in blocking buffer (1:2 dilution). After extensive washes in blocking buffer, the cells were incubated for 30 min with the fluorescent secondary antibody (anti-mouse IgG FITC; Sigma) diluted in blocking buffer (1:200 dilution). The coverslips were again washed extensively in blocking buffer and once in PBS, mounted in Tris-HCl (0.5 M, pH 8.5) containing 70% glycerol, and observed under a Leica DMRB microscope equipped for epifluorescence. The functionality of the expressed protein was investigated by incubating the transfected COS-7 cells with different fatty acid albuminic complexes. Each fatty acid was saponified by incubation for 30 min at 70°C with 2 M KOH in ethanol. The resulting fatty acid salt was dissolved at pH 10 in DMEM containing 1% (w/v) BSA. After 15 min of sonication followed by 5 h of shaking, the pH was adjusted to 7.3. FCS was added (10%, v/v), and the final fatty acid concentration of the incubation medium was 0.2 mM unless stated otherwise. At 3 h after transfection, the incubation of COS-7 cells was initiated by replacing the culture medium with 20 ml of the fatty acid-containing medium per 10 cm dish. Incubation was performed for 24 h at 37°C in 5% CO2 atmosphere. COS-7 cells were washed twice with ice-cold PBS (150 mM NaCl and 5 mM Na phosphate, pH 7.4) and scraped into PBS. After centrifugation, the cell pellet was resuspended in PBS and sonicated at 20 W for 5 s. The protein content of the cell homogenate was determined by a modified Lowry procedure (21Bensadoun A. Weinstein D. Assay of proteins in the presence of interfering materials.Anal. Biochem. 1976; 70: 241-250Crossref PubMed Scopus (2733) Google Scholar). Cellular lipids were extracted with hexane-isopropanol (3:2; v/v) as described previously (22Rioux V. Lemarchal P. Legrand P. Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes.J. Nutr. Biochem. 2001; 11: 198-207Crossref Scopus (57) Google Scholar). After saponification, fatty acids were methylated with boron trifluoride (14% in methanol) at 70°C for 30 min. Fatty acid methyl esters were extracted with pentane and analyzed by gas chromatography using an Agilent Technologies 6890N (Bios Analytique, Toulouse, France) with a split injector (1:20) at 250°C and a bonded silica capillary column (30 m × 0.25 mm internal diameter; BPX 70; SGE, Villeneuve-St-Georges, France) with a stationary phase of 70% cyanopropylpolysilphenylene-siloxane (0.25 μm film thickness). Helium was used as the gas vector (average velocity, 24 cm/s). The column temperature program started at 150°C, was ramped at 2°C/min to 220°C, and was held at 220°C for 10 min. The flame ionization detector temperature was 250°C. Identification of fatty acid methyl ester peaks was based on retention times obtained for methyl esters prepared from fatty acid standards. Cell homogenates were prepared as described above at 48 h after transfection. Desaturase activity was assayed in a 1 ml mixture containing 100 μl of cell homogenate (5–8 mg protein/ml), 150 mM phosphate buffer (pH 7.2), 6 mM MgCl2, 7.2 mM ATP, 0.54 mM CoA, and 0.8 mM NADH. The reaction was started by adding 60 nmol of [1-14C]18:3n-3 (52 mCi/mmol) and stopped with 1 ml of 2 M KOH in ethanol after 1 h of incubation at 37°C. To assess the substrate quality, a control assay was also run by stopping the reaction before adding the substrate. Fatty acid saponification was performed at 70°C for 30 min. After acidification, the fatty acids were extracted with diethylether, converted to fatty acid naphthacyl esters, and separated by HPLC as described previously (23Rioux V. Catheline D. Bouriel M. Legrand P. High performance liquid chromatography of fatty acids as naphthacyl esters.Analysis. 2000; 27: 186-193Google Scholar). Collected fractions were subjected to liquid scintillation counting (Packard Tri-Carb 1600 TR, Meriden, CT). Desaturase activities were normalized for transfection efficiency by measuring the β-galactosidase activity corresponding to 3 μg of a cotransfected β-galactosidase-expressing vector (pCMV/β-Gal). The β-galactosidase activity was assayed at 37°C in 20 μl of lysate mixed with 142 μl of 0.1 M phosphate buffer (pH 7.5), 2.5 μl of 0.1 M MgCl2, 4.5 M β-mercaptoethanol, and 55 μl of O-nitrophenyl β-d-galactopyranoside (Sigma) (4 mg/ml in 0.1 M phosphate buffer). Two days after transfection, cells were lysed at 4°C in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mM PMSF, and 10 μg/ml aprotinin). Lysates were subjected to 12 h of immunoprecipitation with 1 μg of either monoclonal anti-Flag M2 (Sigma) or polyclonal anti-HA Y11 (Santa Cruz Biotechnologies, Le Perray-en-Yvelines, France) followed by adsorption to Sepharose-coupled protein G (Sigma) for 3 h. Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting. For determination of total protein levels, aliquots of cell lysates were also subjected to direct immunoblotting. Reduced protein samples were analyzed by SDS-PAGE and blotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). To measure wild-type and mutant Δ6-desaturase expression, the anti Δ6-desaturase serum S1 targeting the C-terminal region of the protein was used at a 1:2,000 dilution, as described previously (13D'Andrea S. Guillou H. Jan S. Catheline D. Thibault J-N. Bouriel M. Rioux V. Legrand P. The same rat delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis.Biochem. J. 2002; 364: 49-55Crossref PubMed Scopus (96) Google Scholar). To measure cytochrome b5 expression, anti-cytochrome b5 (24Borgese N. Gazzoni I. Barberi M. Colombo S. Pedrazzini E. Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but competing pathways.Mol. Biol. Cell. 2001; 12: 2482-2496Crossref PubMed Scopus (104) Google Scholar) was used at a 1:200 dilution. Anti-HA was used at a 1:200 dilution, anti-Flag was used at a 1:300 dilution, and anti-actin (Sigma) was used at a 1:100 dilution. The secondary antibody was a peroxidase-conjugated anti-rabbit IgG (Sigma) or a peroxidase-conjugated anti-mouse IgG (Sigma). Saturation and incubation with antibodies were performed for 90 min in TBS (20 mM Tris-HCl and 150 mM NaCl, pH 7.4) containing 0.05% Tween-20 and 5% nonfat dry milk. Washes were performed in TBS containing 0.05% Tween-20. Peroxidase activity was revealed using ECL Plus reagent according to the manufacturer's instructions (Amersham Biosciences, Uppsala, Sweden) and scanned with the Molecular Dynamics Storm (Amersham Biosciences). A C-terminally myc-tagged rat Δ6-desaturase was expressed in COS-7 cells. Myc-tagged Δ6-desaturase has a molecular mass of 47 kDa as detected by Western blot using a serum targeting rat Δ6-desaturase (Fig. 1A). Using anti-myc antibody, we determined the subcellular localization of C-terminally myc-tagged rat Δ6-desaturase expressed in COS-7 cells. In transiently transformed COS-7 cells, a perinuclear network was observed (Fig. 1B). This typical pattern suggests that rat Δ6-desaturase localizes in the endoplasmic reticulum. The wild-type (Δ6) and the three mutated forms of rat Δ6-desaturases with deletion of the 53HPGG56 motif (Δ6−53HPGG56), deletion of H53 (Δ6−H53), or substitution of an alanine for H53 (Δ6−H53A) expressed in COS-7 cells were analyzed by Western blotting. A serum targeting the C-terminal region of the protein (13D'Andrea S. Guillou H. Jan S. Catheline D. Thibault J-N. Bouriel M. Rioux V. Legrand P. The same rat delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis.Biochem. J. 2002; 364: 49-55Crossref PubMed Scopus (96) Google Scholar), which has not been modified by site-directed mutagenesis, was used to probe the membranes. Western blotting revealed that the wild type and the three mutated forms of rat Δ6-desaturase were similarly expressed in COS-7 cells (Fig. 2). The deletion of the 53HPGG56 motif or of H53, or the substitution of an alanine for H53 did not alter rat Δ6-desaturase expression in this cell line. The in vitro Δ6-desaturase assay performed on the COS-7 cell lysates corresponding to the samples used for Western blotting showed a dramatic increase in Δ6-desaturase activity only in cells expressing the wild-type enzyme, compared with nontransfected cells (Fig. 2). The deletion of the 53HPGG56 motif or of H53, or the substitution of an alanine for H53 suppressed the activity of Δ6-desaturase as measured in vitro. We analyzed the fatty acid profiles of COS-7 cells transfected or not with pCMV/Δ6, pCMV/Δ6−53HPGG56, pCMV/Δ6−H53, or pCMV/Δ6−H53A and incubated or not with C18:3n-3 for 24 h (Fig. 3). In each case, the presence of recombinant desaturases was assessed by Western blotting (data not shown). The presence of C20:3n-3 in cells incubated with C18:3n-3 shows that these cells have incorporated and elongated the C18:3n-3 (Fig. 3B–F). In COS-7 cells that have not been incubated with C18:3n-3, the presence of C20:3n-3 was not detected (Fig. 3A). Two additional peaks were observed in cells expressing the wild-type rat Δ6-desaturase (Fig. 3C), whereas these peaks were absent in nontransfected COS-7 cells (Fig. 3A, B) and in cells expressing the mutated forms of Δ6-desaturase (Fig. 3D–F). These two additional fatty acids were identified as C18:4n-3, produced by Δ6-desaturation of C18:3n-3, and C20:4n-3, which corresponds to newly synthesized C18:4n-3 subsequently elongated in COS-7 cells. Together with our in vitro analyses, these results indicate that only the wild-type form of Δ6-desaturase confers to this cell line the capacity of C18:3n-3 Δ6-desaturation. We first used α-linolenic acid (C18:3n-3) for functional analysis of wild-type and mutated forms of Δ6-desaturase. However, it has been shown that the gene encoding animal Δ6-desaturase (FADS2) encodes a protein that acts not only on 18-carbon polyunsaturated substrates (4Cho H.P. Nakamura M.T. Clarke S.D. Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase.J. Biol. Chem. 1999; 274: 471-477Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar, 5Aki T. Shimada Y. Inagaki K. Higashimoto H. Kawamoto S. Shigeta S. Ono K. Suzuki O. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase.Biochem. Biophys. Res. Commun. 1999; 255: 575-579Crossref PubMed Scopus (111) Google Scholar) but also on 24-carbon PUFA (13D'Andrea S. Guillou H. Jan S. Catheline D. Thibault J-N. Bouriel M. Rioux V. Legrand P. The same rat delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis.Biochem. J. 2002; 364: 49-55Crossref PubMed Scopus (96) Google Scholar, 14de Antueno R.J. Knickle L.C. Smith H. Elliot M.L. Allen S.J. Nwaka S. Winther M.D. Activity of human delta5 and delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.FEBS Lett. 2001; 509: 77-80Crossref PubMed Scopus (99) Google Scholar) and palmitic acid (15Guillou H. Rioux V. Catheline D. Thibault J-N. Bouriel M. Jan S. D'Andrea S. Legrand P. Conversion of hexadecanoic acid to hexadecenoic acid by rat delta6-desaturase.J. Lipid Res. 2003; 44: 450-454Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, we investigated the activity of the wild-type and mutated forms of Δ6-desaturases in several other substrates. For this, COS-7 cells transfected with pCMV/Δ6, pCMV/Δ6−53HPGG56, pCMV/Δ6−H53, or pCMV/Δ6−H53A were incubated in the presence of palmitic acid (C16:0), linoleic acid (C18:2n-6), or docosapentaenoic acid (C22:5n-3). After 24 h of incubation, the cellular fatty acid methyl ester profiles were analyzed and used to determine the level of Δ6-desaturation. The expression of each form of recombinant desaturase was systematically assessed by Western blotting (data not shown). As shown in Table 2, regardless of which fatty acid was incubated with COS-7 cells, only the wild-type form of rat Δ6-desaturase conferred to the cells the capacity to act on C16:0, C18:2n-6, and C24:5n-3 (produced by cellular elongation of incubated C22:5n-3), compared with nontransfected cells. The level of C16:1n-10, C18:3n-6, and C24:6n-3 detectable in transfected cells expressing mutated forms of Δ6-desaturase was similar to that in nontransfected cells (see supplementary data).TABLE 2Desaturation index of COS-7 cells not transfected (control) or transfected with pCMV/Δ6, pCMV/Δ6−H53, pCMV/Δ6−H53A, or pCMV/Δ6−53HPGG56 and incubated with different Δ6-desaturase substratesIncubated Fatty AcidC16:0C18:2n-6C22:5n-3Δ6-Desaturase substrateC16:0C18:2n-6C24:5n-3Desaturation index (%)C16:1n-10/C16:0 × 100(C18:3n-6 + C20:3n-6)/C18:2n-6 × 100C24:6n-3/C24:5n-3 × 100Transfection Control0.721.27nd pCMV/Δ615.3137.229.58 pCMV/Δ6−H530.782.04nd pCMV/Δ6−H53A0.811.62nd pCMV/Δ6−53HPGG560.552.08ndCOS-7 cells were transfected or not and subsequently cultivated for 24 h with distinct albumin-bound fatty acids (200 μM). Then, the cells were washed extensively with PBS and cellular fatty acids were prepared for gas chromatography analysis as described in Materials and Methods. nd, the Δ6-desaturation product was not detectable. Open table in a new tab COS-7 cells were transfected or not and subsequently cultivated for 24 h with distinct albumin-bound fatty acids (200 μM). Then, the cells were washed extensively with PBS and cellular fatty acids were prepared for gas chromatography analysis as described in Materials and Methods. nd, the Δ6-desaturation product was not detectable. The microsomal form of rat cytochrome b5 was cotransfected with the wild-type or mutated forms of Δ6-desaturase in COS-7 cells. The expression of Δ6-desaturases and cytochrome b5 was controlled by Western blotting (Fig. 4). Then, relative Δ6-desaturase activities were measured in vitro on the cell lysates. This assay showed that overexpression of rat microsomal cytochrome b5 cannot compensate for the effect of the deletions or the mutation in the 53HPGG56 motif of the rat Δ6-desaturase cytochrome b5 domain (Fig. 4). Interestingly, coexpression of rat endoplasmic reticulum cytochrome b5 with wild-type Δ6-desaturase markedly increased (2.2-fold) the Δ6-desaturase activity, compared with Δ6-desaturase alone. The possibility that Δ6-desaturase may interact with cytochrome b5 was investigated in COS-7 cells. Immunoprecipitation of lysates from transfected COS-7 cells with an antibody directed against Flag-tagged Δ6-desaturase revealed the presence of HA-tagged cytochrome b5 (Fig. 5). Similarly, when immunoprecipitation was performed with an antibody directed against HA-tagged cytochrome b5, the Flag-tagged Δ6-desaturase was coimmunoprecipitated. Interestingly, Flag-tagged Δ6-desaturase lacking the HPGG motif also coimmunoprecipitated with HA-tagged cytochrome b5, suggesting that the 53HPGG56 motif of the desaturase is not required for interaction between Δ6-desaturase and cytochrome b5. This study presents the localization in an animal cell line of rat Δ6-desaturase in the endoplasmic reticulum (Fig. 1) and provides the first evidence for the requirement of the 53HPGG56 motif, and at least H53 in this motif, in the cytochrome b5-like domain of this enzyme. As the mutated proteins had no activity when expressed at levels similar to those in the wild type, we concluded that the domain is important for the protein activity but not for its expression or stability (Fig. 2). Moreover, our data showed that this motif, and particularly H53, plays a critical role in Δ6-desaturase activity in its different substrates (Fig. 3, Table 2). This observation is consistent with the previously reported essentialness of H41 in borage Δ6-desaturase (11Sayanova O. Shewry P. Napier J. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.Plant Physiol. 1999; 121: 641-646Crossref PubMed Scopus (63) Google Scholar) and with the role of the cytochrome b5-like domain in yeast Δ9-desaturase activity (12Mitchell A.G. Martin C.E. A novel cytochrome b5-like domain is linked to the carboxyl terminus of the Saccharomyces cerevisiae delta-9 fatty acid desaturase.J. Biol. Chem. 1995; 50: 29766-29772Abstract Full Text Full Text PDF Scopus (131) Google Scholar). The COS-7 cells are unlikely to be deficient in cytochrome b5. For example, rat Δ9-desaturase, which requires cytochrome b5 to function, is active when expressed in COS7 cells (15Guillou H. Rioux V. Catheline D. Thibault J-N. Bouriel M. Jan S. D'Andrea S. Legrand P. Conversion of hexadecanoic acid to hexadecenoic acid by rat delta6-desaturase.J. Lipid Res. 2003; 44: 450-454Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This suggests that endogenous cytochrome b5, constitutively present in COS-7 cells, cannot rescue the activity of a Δ6-desaturase whose cytochrome b5-like domain has been mutated or deleted. To further address this hypothesis, we measured Δ6-desaturase activities in COS-7 cells transiently transfected with distinct forms of Δ6-desaturase in the presence or absence of coexpressed rat cytochrome b5. Similarly, we showed that rat cytochrome b5 coexpressed in this cell line did not rescue the activity of mutated forms of Δ6-desaturase (Fig. 4). Thus, neither endogenous microsomal cytochrome b5 nor coexpressed rat cytochrome b5 could rescue the activity of mutated Δ6-desaturases. The major role of the rat Δ6-desaturase cytochrome b5-like domain may have led to the speculation that the enzyme can function independently of free microsomal cytochrome b5. However, coexpression of microsomal rat cytochrome b5 with rat Δ6-desaturase is necessary for an optimal PUFA desaturation in yeast (17Michinaka Y. Aki T. Inagaki K. Higashimoto H. Shimada Y. Nakajima T. Shimauchi T. Ono K. Suzuki O. Production of polyunsaturated fatty acids by genetic engineering of yeast.J. Oleo Sci. 2001; 5: 359-365Crossref Scopus (5) Google Scholar). Thus, the role of microsomal cytochrome b5 in the process of Δ6-desaturation could not be dismissed. Consistent with this proposal, we showed that microsomal cytochrome b5 stimulated Δ6-desaturase activity when coexpressed in a mammalian cell line (Fig. 4). Because the 53HPGG56 region of the rat Δ6-desaturase cytochrome b5-like domain may represent an important motif for the structure of the protein and its putative interaction with other proteins, we tested whether Δ6-desaturase or Δ6-desaturase with complete deletion of the 53HPGG56 sequence interacts with cytochrome b5 in COS-7 cells. When coexpressed in COS-7 cells, wild-type Δ6-desaturase interacted with cytochrome b5 (Fig. 5). This protein-protein interaction may contribute to the effect of cytochrome b5 on Δ6-desaturase activity. Interestingly, we observed that the complete deletion of the 53HPGG56 motif did not alter the interaction between Δ6-desaturase and cytochrome b5 (Fig. 5), providing evidence that this motif is not necessary for interaction between these two proteins, whereas cytochrome b5 could not rescue the activity of mutated forms of Δ6-desaturases (Fig. 4). Therefore, the different results described here assess the important role of both the Δ6-desaturase cytochrome b5-like domain and the microsomal cytochrome b5 in the process of Δ6-desaturation. This study also shows that microsomal cytochrome b5 cannot compensate for the essential role of the highly conserved 53HPGG56 motif in the rat Δ6-desaturase cytochrome b5-like domain. The precise role of free cytochrome b5 in Δ6-desaturase activity should be further defined. It would be interesting to investigate further the cytochrome b5-Δ6-desaturase interaction and the stimulatory effect of cytochrome b5 using models with greater physiological expression of both proteins. Whether cytochrome b5 contributes to an electron transfer required for Δ6-desaturase activity remains to be elucidated. As has been shown for cytochrome P450 monooxygenase (25Schenkman J.B. Jansson I. The many roles of cytochrome b5.Pharmacol. Ther. 2003; 97: 139-152Crossref PubMed Scopus (376) Google Scholar), the possibility that cytochrome b5 may not function as an electron transfer component in the Δ6-desaturase enzymatic system could be considered. Together, these results suggest essential and distinct roles for free cytochrome b5 and the fused cytochrome b5-like domain in governing Δ6-desaturase activity. The authors thank M. Bouriel, A. Leborgne, and K. Cung for helpful technical assistance. The authors thank Dr. D. Catheline for monitoring GC analysis, C. Cauty for introducing us to the use of the epifluorescence microscope, and Dr. N. Borgese and Dr. A. Atfi for critically reading the manuscript, for helpful discussions, and for the pCMV/HAHA construct and the antibody to cytochrome b5. The C24:6n-3 methyl ester was a kind gift from Dr. K. Ishiara. The anti-myc antibody and the pCMV/β-Gal constructs were generously provided by Dr. S. Suire and Dr. C. Diot, respectively. 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