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• W2023802690 abstract "2-Hydroxysphingolipids are a subset of sphingolipids containing 2-hydroxy fatty acids. The 2-hydroxylation occurs during de novo ceramide synthesis and is catalyzed by fatty acid 2-hydroxylase (also known as fatty acid α-hydroxylase). In mammals, 2-hydroxysphingolipids are present abundantly in brain because the major myelin lipids galactosylceramides and sulfatides contain 2-hydroxy fatty acids. Here we report identification and characterization of a human gene that encodes a fatty acid 2-hydroxylase. Data base searches revealed a human homologue of the yeast ceramide 2-hydroxylase gene (FAH1), which we named FA2H. The FA2H gene encodes a 372-amino acid protein with 36% identity and 46% similarity to yeast Fah1p. The amino acid sequence indicates that FA2H protein contains an N-terminal cytochrome b5 domain and four potential transmembrane domains. FA2H also contains the iron-binding histidine motif conserved among membrane-bound desaturases/hydroxylases. COS7 cells expressing human FA2H contained 3–20-fold higher levels of 2-hydroxyceramides (C16, C18, C24, and C24:1) and 2-hydroxy fatty acids compared with control cells. Microsomal fractions prepared from transfected COS7 cells showed tetracosanoic acid 2-hydroxylase activities in an NADPH- and NADPH: cytochrome P-450 reductase-dependent manner. FA2H lacking the N-terminal cytochrome b domain had little activity, indicating that this domain is 5a functional component of this enzyme. Northern blot analysis showed that the FA2H gene is highly expressed in brain and colon tissues. These results demonstrate that the human FA2H gene encodes a fatty acid 2-hydroxylase. FA2H is likely involved in the formation of myelin 2-hydroxy galactosylceramides and -sulfatides. 2-Hydroxysphingolipids are a subset of sphingolipids containing 2-hydroxy fatty acids. The 2-hydroxylation occurs during de novo ceramide synthesis and is catalyzed by fatty acid 2-hydroxylase (also known as fatty acid α-hydroxylase). In mammals, 2-hydroxysphingolipids are present abundantly in brain because the major myelin lipids galactosylceramides and sulfatides contain 2-hydroxy fatty acids. Here we report identification and characterization of a human gene that encodes a fatty acid 2-hydroxylase. Data base searches revealed a human homologue of the yeast ceramide 2-hydroxylase gene (FAH1), which we named FA2H. The FA2H gene encodes a 372-amino acid protein with 36% identity and 46% similarity to yeast Fah1p. The amino acid sequence indicates that FA2H protein contains an N-terminal cytochrome b5 domain and four potential transmembrane domains. FA2H also contains the iron-binding histidine motif conserved among membrane-bound desaturases/hydroxylases. COS7 cells expressing human FA2H contained 3–20-fold higher levels of 2-hydroxyceramides (C16, C18, C24, and C24:1) and 2-hydroxy fatty acids compared with control cells. Microsomal fractions prepared from transfected COS7 cells showed tetracosanoic acid 2-hydroxylase activities in an NADPH- and NADPH: cytochrome P-450 reductase-dependent manner. FA2H lacking the N-terminal cytochrome b domain had little activity, indicating that this domain is 5a functional component of this enzyme. Northern blot analysis showed that the FA2H gene is highly expressed in brain and colon tissues. These results demonstrate that the human FA2H gene encodes a fatty acid 2-hydroxylase. FA2H is likely involved in the formation of myelin 2-hydroxy galactosylceramides and -sulfatides. Sphingolipids are a large class of lipids found in all eukaryotic cells and are involved in a variety of cellular processes. The structural diversity of sphingolipids stems from over 300 known distinct head groups as well as modifications of the hydrophobic ceramide moiety. One of the common modifications of the ceramide moiety is 2-hydroxylation of the N-acyl chain. Sphingolipids with 2-hydroxy fatty acid are found in most organisms including plants, yeast, worms, vertebrate animals, and some bacterial species. In mammals, 2-hydroxy fatty acid-containing sphingolipids are uniquely abundant in nervous and epidermal tissues. In mammalian central and peripheral nervous systems, galactosylceramides and sulfatides (3-sulfate ester of galactosylceramide) are major lipid components of myelin (1Nonaka G. Kishimoto Y. Biochim. Biophys. Acta. 1979; 572: 432-441Crossref PubMed Scopus (61) Google Scholar, 2Norton W.T. Cammer W. Morell P. Myelin. Plenum Press, New York1984: 147-195Crossref Google Scholar). These glycosphingolipids contain a high proportion (∼50%) of 2-hydroxy fatty acid (3Hoshi M. Williams M. Kishimoto Y. J. Neurochem. 1973; 21: 709-712Crossref PubMed Scopus (52) Google Scholar) and are critical components of myelin (4Stoffel W. Bosio A. Curr. Opin. Neurobiol. 1997; 7: 654-661Crossref PubMed Scopus (114) Google Scholar, 5Coetzee T. Suzuki K. Popko B. Trends Neurosci. 1998; 21: 126-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In mammalian epidermal tissues, there are several unique, very long chain ceramides with 2-hydroxy fatty acids, which are critical for the permeability barrier function of epidermis (6Wertz P.W. Downing D.T. J. Lipid Res. 1983; 24: 759-765Abstract Full Text PDF PubMed Google Scholar, 7Downing D.T. J. Lipid Res. 1992; 33: 301-313Abstract Full Text PDF PubMed Google Scholar). Several biophysical studies (8Pascher I. Sundell S. Chem. Phys. Lipids. 1977; 20: 175-191Crossref Scopus (208) Google Scholar, 9Boggs J.M. Koshy K.M. Rangaraj G. Biochim. Biophys. Acta. 1988; 938: 361-372Crossref PubMed Scopus (50) Google Scholar, 10Lofgren H. Pascher I. Chem. Phys. Lipids. 1977; 20: 273-284Crossref PubMed Scopus (143) Google Scholar) demonstrated that the 2-hydroxyl group in sphingolipids has a profound effect in the lipid organization within membranes because of its hydrogen-bonding capability. The participation of the 2-hydroxyl group in hydrogen bonds with neighboring lipids was shown by analysis of the crystal structure of synthetic glycosphingolipids with 2-hydroxyoctadecanoic acid (8Pascher I. Sundell S. Chem. Phys. Lipids. 1977; 20: 175-191Crossref Scopus (208) Google Scholar) and by analysis of the phase transition temperature (9Boggs J.M. Koshy K.M. Rangaraj G. Biochim. Biophys. Acta. 1988; 938: 361-372Crossref PubMed Scopus (50) Google Scholar). The monolayer behavior of synthetic ceramides showed that the 2-hydroxyl group promotes condensation to a close-packed arrangement (10Lofgren H. Pascher I. Chem. Phys. Lipids. 1977; 20: 273-284Crossref PubMed Scopus (143) Google Scholar). These studies provide a physical basis for the effects of 2-hydroxysphingolipids in biomembranes. The 2-hydroxylation of sphingolipid N-acyl chains occurs during de novo ceramide synthesis and is catalyzed by the enzyme fatty acid 2-hydroxylase (also known as fatty acid α-hydroxylase). Biochemical properties of this enzyme have been studied in rat brain postnuclear (11Hoshi M. Kishimoto Y. J. Biol. Chem. 1973; 248: 4123-4130Abstract Full Text PDF PubMed Google Scholar) or microsomal fractions (12Akanuma H. Kishimoto Y. J. Biol. Chem. 1979; 254: 1050-1060Abstract Full Text PDF PubMed Google Scholar). The rat brain fatty acid 2-hydroxylase requires molecular oxygen, Mg2+, pyridine nucleotides (NADPH or NADH), and a microsomal electron transport system (13Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar, 14Shigematsu H. Hisanari Y. Kishimoto Y. Int. J. Biochem. 1990; 22: 1427-1432Crossref PubMed Scopus (6) Google Scholar). Insensitivity to carbon monoxide indicates that the rat brain fatty acid 2-hydroxylase is not a P-450 enzyme but another type of mixed function oxidase (11Hoshi M. Kishimoto Y. J. Biol. Chem. 1973; 248: 4123-4130Abstract Full Text PDF PubMed Google Scholar). In the yeast Saccharomyces cerevisiae most sphingolipids contain 2-hydroxy fatty acid. The 2-hydroxylation is dependent on the FAH1 (also known as SCS7) gene, which has been identified as a gene containing a cytochrome b5-like sequence (15Mitchell A.G. Martin C.E. J. Biol. Chem. 1997; 272: 28281-28288Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and as a suppressor of the Ca2+-sensitive phenotype of csg2 mutants (16Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Yeast fah1 mutants show increased resistance to pore-forming antifungal agents, presumably because of altered plasma membrane properties (17Hama H. Young D.A. Radding J.A. Ma D. Tang J. Stock S.D. Takemoto J.Y. FEBS Lett. 2000; 478: 26-28Crossref PubMed Scopus (30) Google Scholar). Yeast Fah1p is a member of the membrane-bound desaturase/hydroxylase family with the conserved histidine motif (HX(3–4)HX(7–41)HX(2–3)-HHX(61–189)(H/Q)X(2–3)HH), which is thought to coordinate a non-heme di-iron cluster at an active site (13Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar). The reactions catalyzed by the enzymes in this family require electron donors and molecular oxygen (13Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar). For yeast Fah1p, the terminal electron donor is likely the intramolecular cytochrome b5 domain. It is notable that the predicted properties of yeast Fah1p are consistent with the biochemical properties of rat brain fatty acid 2-hydroxylase. Therefore, it is a reasonable assumption that mammalian fatty acid 2-hydroxylases are encoded by FAH1 homologues. Here we report identification and characterization of the human FA2H gene, which was identified based on the sequence similarity to the yeast FAH1 gene. Fatty acid 2-hydroxylase activity was found in COS7 cells expressing the human FA2H gene. Materials—Fetal bovine serum was purchased from Atlanta Biologicals (Norcross, GA). [α-32P]dCTP was purchased from PerkinElmer Life Sciences. Deuterated (3,3,5,5-D4)-tetracosanoic acid was purchased from Larodan Fine Chemicals (Malmö, Sweden). A synthetic peptide corresponding to the C-terminal 20 amino acids of FA2H (NH2-KLWDYCFHTLTPEKPHLKTQ-COOH) was synthesized by AnaSpec (San Jose, CA). Anti-hFA2H polyclonal antibodies for the C-terminal peptide were generated in rabbits and affinity purified using immobilized antigen. Horseradish peroxidase (HRP) 1The abbreviations used are: HRP, horseradish peroxidase; GC/MS, gas chromatography/mass spectrometry.-linked anti-FLAG M2 monoclonal antibodies (A8592) were purchased from Sigma. HRP-linked sheep anti-mouse IgG (NXA931), HRP-linked donkey anti-rabbit IgG (NA934), and the ECL Western blotting detection reagents were purchased from Amersham Biosciences. Goat anti-rabbit cytochrome b5 antibodies (RDI-CYTOB5abG) and HRP-linked donkey anti-goat IgG antibodies (RDI-705035147) were purchased from Research Diagnostics, Inc. (Flanders, NJ). Purified human cytochrome b5 was purchased from PanVera (Madison, WI). Purified human NADPH:P-450 reductase and NADPH regenerating system solutions were purchased from BD Biosciences. Cell Culture—COS7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose and l-glutamine, sodium pyruvate, and 10% fetal bovine serum. Cells were maintained at 5% CO2 at 37 °C. Human FA2H Expression Plasmid—A human gene with significant similarity to the yeast FAH1 gene was identified by BLAST analyses. A human FA2H cDNA clone was purchased from the American Type Culture Collection (ATCC no. MGC-10804). The original data base entry indicated the open reading frame starting at the methionine 93 (Fig. 1). The FA2H open reading frame was amplified accordingly by PCR using a pair of oligonucleotides (5′-GCAGGGATCCATGGAGAACGAGCCTGTAGC-3′ and 5′-TGGGAATTCTCACTGCGTCTTCAGGTGGGG-3′), which were digested with BamHI and EcoRI and cloned into pBluescript II vector, resulting in pBS-hFA2HΔN. Later, a sequence comparison among the human, mouse, and rat homologues revealed an N-terminal extension of 92 amino acids corresponding to a cytochrome b5 domain. To create a full-length recombinant human FA2H containing the N-terminal cytochrome b5 domain, a 276-bp NcoI fragment corresponding to the N-terminal 92 amino acids was inserted into the NcoI site of pBS-hFA2HΔN, resulting in pBS-hFA2H. Following sequence verification, the two versions of FA2H (hFA2HΔN and hFA2H) were subcloned into the mammalian expression vector pcDNA3 (Invitrogen), resulting in pcDNA3-hFA2HΔN and pcDNA3-hFA2H, respectively. To introduce an N-terminal FLAG tag, the BamHI-EcoRI fragment of pBS-hFA2H was subcloned into pYES2-FLAG (18Mao C. Xu R. Szulc Z.M. Bielawski J. Becker K.P. Bielawska A. Galadari S.H. Hu W. Obeid L.M. J. Biol. Chem. 2003; 278: 31184-31191Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), resulting in pYES2-FLAG-hFA2H. The FLAG-hFA2H fragment was cut out with HindIII and EcoRI and subcloned into pcDNA3, resulting in pcDNA3-FLAG-hFA2H. Transfection—COS7 cells were transfected with pcDNA3, pcDNA3-hFA2H, pcDNA3-hFA2HΔN or pcDNA3-FLAG-hFA2H using FuGENE 6 transfection reagent (Roche Applied Science). Cells were harvested 24 h after transfection by trypsin-EDTA treatment for lipid analyses or were directly lysed in 6-well culture dishes for Western blot analyses. Cell Fractionation—COS7 cells were transfected in 6-well culture dishes. Twenty-four hours after transfection, cells were harvested by trypsin-EDTA treatment, resuspended in a solution of 10 mm Tris-HCl, pH 7.4, 1% glycerol, and 1 mm phenylmethylsulfonyl fluoride, and lysed by freezing-thawing followed by sonication (amplitude 29%, 5 s on, 0.1 s off, 12 cycles) (Fisher Sonic Dismembrator Model 500). Cell lysate was centrifuged at 1000 × g for 5 min to remove unbroken cells and large debris. The supernatant (low speed supernatant) was centrifuged at 100,000 × g for 2 h to separate the supernatant (cytosolic fraction) and the pellet (membrane fraction). Western Blot Analysis—COS7 cells were transfected in 6-well culture dishes. Twenty-four hours after transfection, cells were washed twice with phosphate-buffered saline, lysed with 100 μl of SDS-PAGE lysis buffer/well (10 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 150 mm NaCl, 25 μg/ml phenylmethylsulfonyl fluoride), and mixed with an equal volume of 2× SDS-PAGE sample buffer. Following electrophoresis, proteins were blotted onto nitrocellulose membranes and incubated with HRP-linked anti-FLAG M2 monoclonal antibodies (1:500) or anti-human FA2H polyclonal antibodies (1:1000) followed by incubation with HRP-linked donkey anti-rabbit IgG (1:5000). Protein bands were detected using ECL Western blotting detection reagents. For detection of cytochrome b5, goat anti-rabbit cytochrome b5 antibodies and HRP-linked donkey anti-goat IgG antibodies were used. Purified human cytochrome b5 was used as a positive control. Ceramide Determination by Liquid Chromatography/Tandem Mass Spectrometry—Approximately 1 × 106 COS7 cells were transfected with pcDNA3 or pcDNA3-hFA2H, harvested by trypsin-EDTA treatment, and washed with Hanks' balanced salt solution and phosphate-buffered saline. Internal standards were added to the cells, and lipids were extracted with ethyl acetate/isopropanol/water (60:30:10, v/v). A small aliquot of each lipid extract was set aside for phosphate quantitation. Lipid extracts were evaporated to dryness and reconstituted in 100 μl of methanol. The reconstituted samples were injected onto a Thermo Finnigan Surveyor liquid chromatography system with a BDS Hypersil C8 column (150 × 3.2 mm, 3-μm particle size) and eluted with a mobile phase consisting of 1 mm methanolic ammonium formate and 2 mm aqueous ammonium formate. Electrospray ionization-tandem mass spectroscopic analysis of ceramides was performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring positive ionization mode. Peaks of the target analytes and internal standards were collected and processed using the Xcalibur software system. Calibration curves were constructed by plotting peak area ratios of the target analyte to their respective internal standard against concentration using a linear regression model. Lipid phosphates were determined by the method of Van Veldhoven and Bell (19Van Veldhoven P.P. Bell R.M. Biochim. Biophys. Acta. 1988; 959: 185-196Crossref PubMed Scopus (137) Google Scholar). Ceramide contents were normalized to the lipid phosphate. Fatty Acid Determination by Gas Chromatography/Mass Spectrometry—COS7 cells were transfected with pcDNA3 or pcDNA3-hFA2H, harvested, and lysed as described above. Crude cell lysate (0.5 ml) was mixed with a set of internal standards (C15, C17, C19, C21, and C23 fatty acids), and free fatty acids were extracted three times with 1 ml of diethyl ether. Combined diethyl ether extracts were dried under nitrogen. To prepare fatty acid methyl esters, 1 ml of anhydrous methanolic HCl was added to each sample and incubated at 65 °C for 45 min, and samples were dried under nitrogen. To prepare trimethylsilyl derivatives of hydroxyl groups, 100 μl of Tri-Sil reagent (Pierce) was added to each sample and incubated for 30 min at room temperature. One to 2 μl of each sample was applied to a Hewlett-Packard 5890 gas chromatograph with a Restek RTX-5 column (5% diphenyl, 95% dimethyl polysiloxane, 0.25 mm inner diameter, 0.25 μm D.F., 30 m). The injection port and the transfer line were maintained at 250 °C, and the oven temperature was increased from 110 to 300 °C at 10 °C/min. Mass spectra data were obtained on a VG-70S magnetic sector mass spectrometer. Peaks of the target analytes and internal standards were collected and processed using the Opus software system (Micromass Information Systems, Modesto, CA). Calibration curves were constructed by plotting peak area ratios of the target analytes to their respective internal standard against concentration, using linear regression analysis. Fatty Acid 2-Hydroxylase Assay—Membrane fractions were prepared as described under “Cell Fractionation.” Membrane pellets were resuspended in 1 ml of the lysis buffer by brief sonication in a bath sonicator. The assay mixture contained microsomal fractions (100 μg of proteins), 2.7 mm Tris-HCl, pH 7.6, 1.28 mm NADP+, 3.3 mm glucose 6-phosphate, 3.3 mm MgCl2, 0.2 unit of glucose 6-phosphate dehydrogenase, 1 μg of human NADPH:cytochrome P-450 reductase, and 1 μg (2.7 nmol) of (3,3,5,5-D4)-tetracosanoic acid (stock solution was prepared as 10 μg/ml in 1.5 mm α-cyclodextrin) in a total volume of 1.5 ml. The assay mixture was placed in a 50-ml polypropylene tube and incubated at 37 °C for 2 h with shaking (100 rpm) to facilitate the diffusion of oxygen. At the end of incubation, 50 pmol of C23 fatty acid (internal standard) was added to each sample, and fatty acids were extracted three times with 2 ml diethyl ether. The combined diethyl ether extracts were brought to dryness under a stream of nitrogen. Fatty acids were derivatized and quantified as described under “Fatty Acid Determination by GC/MS.” The ions monitored for 2-hydroxy-(3,3,5,5-D4)-tetracosanoic acid had a mass of 415 and 459, corresponding to M-15 and M-59, respectively. The activities were calculated as picomoles of 2-hydroxy-(3,3,5,5-D4)-tetracosanoic acid/mg of protein/min. Northern Blot Analysis—Premade Northern blots containing poly(A)+ RNA from human tissues (Clontech) were probed with FA2H cDNA or a human β-actin control cDNA. The FA2H cDNA probe was a 862-bp fragment obtained by PCR with oligonucleotides FA2H-FBam (5′-GCAGGGATCCATGGAGAACGAGCCTGTAGC-3′) and FA2H-RR1 (5′-TGGGAATTCTCACTGCGTCTTCAGGTGGGG-3′). Probes were labeled with [α-32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences). Hybridization was performed according to the manufacturer's instructions. Identification of Human FA2H Gene—BLAST sequence analyses identified several human cDNA sequences that had significant similarity to yeast FAH1 (also known as SCS7), a gene required for 2-hydroxylation of sphingolipid-associated very long chain fatty acids (15Mitchell A.G. Martin C.E. J. Biol. Chem. 1997; 272: 28281-28288Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 20Dunn T.M. Haak D. Monaghan E. Beeler T.J. Yeast. 1998; 14: 311-321Crossref PubMed Scopus (65) Google Scholar). All the cDNA clones were derived from the same gene located in the human chromosome 16 (NCBI locus identification, 79152), which we named FA2H for fatty acid 2-hydroxylase. The FA2H gene product is a 372-amino acid protein (42.8 kDa) that has 36% identity and 46% similarity to yeast Fah1p (Fig. 1). Yeast Fah1p contains an N-terminal cytochrome b5 domain (NCBI conserved domain data base accession no. 22935), four potential transmembrane domains, and the characteristic histidine motif conserved among membrane-bound desaturases/hydroxylases (consensus: HX(3–4)HX(7–41)HX(2–3)-HHX(61–189)(H/Q)X(2–3)HH) (13Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar, 15Mitchell A.G. Martin C.E. J. Biol. Chem. 1997; 272: 28281-28288Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20Dunn T.M. Haak D. Monaghan E. Beeler T.J. Yeast. 1998; 14: 311-321Crossref PubMed Scopus (65) Google Scholar). These histidines are thought to coordinate the non-heme di-iron cluster at an active site (13Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar). Similarly, human FA2H protein contains a cytochrome b5 domain at its N terminus (amino acids 1–92). Within this domain is the conserved heme-binding domain (His-Pro-Gly-Gly), suggesting that it is functional as an electron carrier. The cytochrome b5 domains of yeast Fah1p and human FA2H lack a membrane anchor, which is present in the microsomal cytochrome b5. The C-terminal three quarters of FA2H are highly homologous to the sphingolipid fatty acid hydroxylase domain (NCBI conserved domain data base accession no. 9675). This domain is found in previously identified FAH1 homologues in Schizosaccharomyces pombe, Arabidopsis thaliana, and Caenorhabditis elegans (15Mitchell A.G. Martin C.E. J. Biol. Chem. 1997; 272: 28281-28288Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), as well as in hypothetical proteins in the fungi Magnaporthe grisea and Neurospora crassa, the malaria mosquito Anopheles gambiae, Drosophila melanogaster, the nematode C. briggsae, Xenopus laevis, Mus musculus, and Rattus norvegicus (NCBI conserved domain architecture retrieval tool). All of these proteins contain the eight conserved histidines (Fig. 1, consensus sequence asterisks) and an N-terminal cytochrome b5 domain with the exception of the A. thaliana homologue, which lacks a cytochrome b5 domain (15Mitchell A.G. Martin C.E. J. Biol. Chem. 1997; 272: 28281-28288Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 2-Hydroxyceramides and 2-Hydroxy Fatty Acids in COS7 Cells Expressing Human FA2H—To show that human FA2H is involved in the formation of 2-hydroxysphingolipids, the levels of ceramides and 2-hydroxyceramides in COS7 cells transfected with pcDNA3 or pcDNA3-hFA2H were determined. Total cellular lipids were extracted 24 h after transfection, and ceramides were fractionated on a reversed-phase high pressure liquid chromatography column and quantified by a tandem mass spectrometer. Fig. 2 shows the levels of ceramides and 2-hydroxyceramides with five different fatty acid chain lengths. Ceramides with normal (non-hydroxy) fatty acids are comparable with or slightly decreased in FA2H-expressing cells compared with the control cells (Fig. 2A). Cellular 2-hydroxyceramides in FA2H-transfected cells increased 3–20-fold, except for 2-hydroxyceramide C20:0 (Fig. 2B). The levels of 2-hydroxyceramides continued to increase after 24 h and reached as much as 40-fold over control cells in 72 h in the case of C16:0 ceramide (data not shown). These results provide evidence that FA2H is involved in the formation of 2-hydroxysphingolipids. The fatty acid 2-hydroxylation is believed to occur prior to ceramide synthesis in mammals. However, it has not been clearly demonstrated whether 2-hydroxylation occurs on free fatty acids. To determine whether 2-hydroxylation occurs on free fatty acids, we measured free 2-hydroxy fatty acids in control and FA2H-transfected cells along with non-hydroxy fatty acids. Because free 2-hydroxy fatty acids were expected to be minor species, a high-resolution GC/MS assay was developed for the quantification of the fatty acids. 2-Hydroxy fatty acids were derivatized as trimethylsilyl ether of methyl esters. Fig. 3 shows the mass spectrum of trimethylsilyl ether of 2-hydroxytetracosanoic acid methyl ester. Electron impact ionization generated two distinct ions with masses of 411 and 455 (Fig. 3, inset). The limit of detection by this method was at the femtomol level. Using this method, non-hydroxy and 2-hydroxy fatty acids were quantified in transfected COS7 cells. As shown in Fig. 4A, non-hydroxy fatty acid levels were indistinguishable between control and FA2H-transfected cells. Consistent with the presence of 2-hydroxyceramides shown in Fig. 2, low levels of 2-hydroxy fatty acids were present in control cells. Among the six different free 2-hydroxy fatty acids analyzed, the levels were 2.3–5.4-fold higher in FA2H-transfected cells over control cells, except for C20 fatty acid (1.5-fold). The highly elevated free 2-hydroxy fatty acid levels are consistent with the possibility that FA2H-dependent 2-hydroxylation occurs on free fatty acids.Fig. 4Fatty acids and 2-hydroxy fatty acids in transfected COS7 cells. Fatty acids were extracted from COS7 cell transfected with pcDNA3 (open bars) or pcDNA3-hFA2H (filled bars). Non-hydroxy fatty acids (A) and 2-hydroxy fatty acids (B) were quantified by GC/MS as described under “Experimental Procedures.” The average and standard deviation of three measurements are shown.View Large Image Figure ViewerDownload (PPT) Western Blot Analysis—The amino acid sequence of human FA2H indicated that it was a membrane-bound, 43-kDa protein. There was another potential translation initiation site at methionine 93, which had been noted as a translation initiation site in some data base entries for the same gene. Interestingly, methionine 93 is located between the N-terminal cytochrome b5 domain and the sphingolipid fatty acid hydroxylase domain. It is conceivable that translation initiation at methionine 93 produces a catalytically active 33-kDa protein, based on the fact that the Arabidopsis homologue, lacking a cytochrome b5 domain, is active when expressed in yeast (15Mitchell A.G. Martin C.E. J. Biol. Chem. 1997; 272: 28281-28288Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To test whether the cytochrome b5 domain is necessary for catalytic activity of recombinant human FA2H, full-length and truncated FA2H, starting from methionine 1 and methionine 93, respectively, were constructed. FLAG-tagged FA2H was also constructed to confirm the presence of the N-terminal portion with anti-FLAG antibodies. When expressed in COS7 cells, the three versions of FA2H were 43, 33, and 44 kDa, as expected (Fig. 5A). There was no detectable endogenous FA2H in COS7 cells, indicating low abundance of endogenous FA2H in these cells and/or poor conservation of the C-terminal portion of FA2H, which was used to generate the antibodies. Western blot with anti-FLAG monoclonal antibodies detected FLAG-tagged FA2H, confirming that the entire N-terminal portion is present in this construct (Fig. 5B). The overexpressed FA2H was exclusively fractionated in the membrane fraction (Fig. 5C) as expected from the presence of four putative transmembrane domains. Anticytochrome b5 antibodies did not cross-react with the full-length human FA2H expressed in COS7 cells (data not shown). In Vitro Fatty Acid 2-Hydroxylase Activity—To show that FA2H-dependent 2-hydroxylation occurs on free fatty acids, we have developed a sensitive and specific in vitro assay using GC/MS for the detection of reaction products. Based on previous studies (14Shigematsu H. Hisanari Y. Kishimoto Y. Int. J. Biochem. 1990; 22: 1427-1432Crossref PubMed Scopus (6) Google Scholar, 21Shigematsu H. Kishimoto Y. Int. J. Biochem. 1987; 19: 41-46Crossref PubMed Scopus (9) Google Scholar) of the rat brain enzyme, fatty acid 2-hydroxylation is coupled with a microsomal electron transport system involving NADH:cytochrome b5 reductase or NADPH: cytochrome P-450 reductase. The requirement of an electron transport system is also predicted from the presence of the cytochrome b5 domain in the human FA2H protein. Because NADPH is a more effective electron donor than NADH for the rat brain enzyme (21Shigematsu H. Kishimoto Y. Int. J. Biochem. 1987; 19: 41-46Crossref PubMed Scopus (9) Google Scholar), our assay system included purified recombinant human NADPH:cytochrome P-450 reductase and an NADPH regeneration system (NADP+, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase). With these components, electron transport would occur in the following sequence: NADPH → NADPH:cytochrome P-450 reductase → cytochrome b5 domain of FA2H → the catalytic di-iron of FA2H. Because human FA2H is a membrane-bound protein as shown above, microsomal fractions of transfected COS7 cells were used as enzyme sources. To distinguish exogenously added substrate from endogenous fatty acids present in microsomal fractions, we used a deuterated fatt" @default.
• W2023802690 created "2016-06-24" @default.
• W2023802690 creator A5000369630 @default.
• W2023802690 creator A5016143807 @default.
• W2023802690 creator A5031897496 @default.
• W2023802690 creator A5052979324 @default.
• W2023802690 creator A5076524129 @default.
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• W2023802690 date "2004-11-01" @default.
• W2023802690 modified "2023-10-16" @default.
• W2023802690 title "The Human FA2H Gene Encodes a Fatty Acid 2-Hydroxylase" @default.
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