The Acyl-CoA Synthetase “Bubblegum” (Lipidosin)
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m310075200
ISSN1083-351X
AutoresZhengtong Pei, Nadia A. Oey, Maartje M. Zuidervaart, Zhenzhen Jia, Yuanyuan Li, Steven J. Steinberg, Kirby D. Smith, Paul A. Watkins,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoAcyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant “bubblegum,” belongs to a previously unidentified enzyme family and is capable of activating both long- and very long-chain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30-35% decrease in activation and β-oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells. Acyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant “bubblegum,” belongs to a previously unidentified enzyme family and is capable of activating both long- and very long-chain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30-35% decrease in activation and β-oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells. Acyl-CoA synthetases (ACS) 1The abbreviations used are: ACSacyl-CoA synthetasehhumanmmouseVLCFAvery long-chain fatty acidsLCFAlong-chain fatty acidsMBPmaltose-binding proteinLACSlong-chain ACSMAMMitochondria-associated membraneMnSODmagnesium superoxide dismutasehCGhuman chorionic gonadotropinmSAmouse serum albuminVLACSvery long-chain ACSXALDX-linked adrenoleukidystrophysiRNAsmall interfering RNABG“bubblegum.”1The abbreviations used are: ACSacyl-CoA synthetasehhumanmmouseVLCFAvery long-chain fatty acidsLCFAlong-chain fatty acidsMBPmaltose-binding proteinLACSlong-chain ACSMAMMitochondria-associated membraneMnSODmagnesium superoxide dismutasehCGhuman chorionic gonadotropinmSAmouse serum albuminVLACSvery long-chain ACSXALDX-linked adrenoleukidystrophysiRNAsmall interfering RNABG“bubblegum.” occupy a central position in fatty acid metabolism. Activation of fatty acids to their CoA derivatives by these enzymes is generally required for further metabolism, whether it be anabolic (e.g. triglyceride, phospholipid, and cholesterol ester synthesis, or chain elongation), catabolic (e.g. β-oxidation in either mitochondria or peroxisomes), or regulatory (e.g. protein acylation) (1Watkins P.A. Prog. Lipid. Res. 1997; 36: 55-83Crossref PubMed Scopus (153) Google Scholar). Numerous enzymes with ACS activity have been described. All contain an AMP-binding domain (Prosite PS00455; us.expasy.org/prosite/) (2Babbitt P.C. Kenyon G.L. Martin B.M. Charest H. Slyvestre M. Scholten J.D. Chang K.H. Liang P.H. Dunawaymariano D. Biochemistry. 1992; 31: 5594-5604Crossref PubMed Scopus (187) Google Scholar) and a second highly conserved motif (3Watkins P.A. Pevsner J. Steinberg S.J. Prostaglandins Leukotrienes Essent. Fatty Acids. 1999; 60: 323-328Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 4Black P.N. Zhang Q. Weimar J.D. DiRusso C.C. J. Biol. Chem. 1997; 272: 4896-4903Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Using these motifs, we classified ACSs into several distinct families, each comprised of multiple members within a given species (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Although most ACSs differ in their tissue distribution, subcellular location, and substrate specificity, information on the specific role in metabolism of each individual enzyme is generally lacking.Min and Benzer (6Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar) described a Drosophila melanogaster mutant, “bubblegum,” that was characterized by neurodegeneration and elevated levels of saturated very long-chain fatty acids (VLCFA) (6Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar). The protein encoded by the gene defective in this mutant had amino acid sequence homology to other ACSs, but inspection of the region containing the second conserved motif revealed that it belonged to a previously unreported ACS family (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Examination of genomic and expressed sequence tag data bases suggests that the “bubblegum” ACS families in humans, mice, and fruitflies contain only two members. 2Z. Pei, Z. Jia, and P. A. Watkins, manuscript in preparation.2Z. Pei, Z. Jia, and P. A. Watkins, manuscript in preparation. Full-length cDNA encoding BG1 3The “bubblegum” (lipidosin) family contains two members. We refer to the enzyme originally described in Drosophila and its mammalian homologs as BG1.3The “bubblegum” (lipidosin) family contains two members. We refer to the enzyme originally described in Drosophila and its mammalian homologs as BG1. was cloned from Drosophila (6Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar), human (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), mouse (7Moriya-Sato A. Hida A. Inagawa-Ogashiwa M. Wada M.R. Sugiyama K. Shimizu J. Yabuki T. Seyama Y. Hashimoto N. Biochem. Biophys. Res. Commun. 2000; 279: 62-68Crossref PubMed Scopus (22) Google Scholar), and rat (8Tang P.Z. Tsai-Morris C.H. Dufau M.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6581-6586Crossref PubMed Scopus (25) Google Scholar) sources. Moriya-Sato et al. (7Moriya-Sato A. Hida A. Inagawa-Ogashiwa M. Wada M.R. Sugiyama K. Shimizu J. Yabuki T. Seyama Y. Hashimoto N. Biochem. Biophys. Res. Commun. 2000; 279: 62-68Crossref PubMed Scopus (22) Google Scholar) proposed the name “lipidosin” for this enzyme.We reported that human BG1 (hBG1) when expressed in COS-1 cells activated both long-chain fatty acids (LCFA) and VLCFA (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The latter activity functionally classifies hBG1 as a very long-chain ACS (VLACS). Moriya-Sato et al. (7Moriya-Sato A. Hida A. Inagawa-Ogashiwa M. Wada M.R. Sugiyama K. Shimizu J. Yabuki T. Seyama Y. Hashimoto N. Biochem. Biophys. Res. Commun. 2000; 279: 62-68Crossref PubMed Scopus (22) Google Scholar) reported that mouse BG1 (mBG1) was expressed in brain, adrenal gland, and testis (7Moriya-Sato A. Hida A. Inagawa-Ogashiwa M. Wada M.R. Sugiyama K. Shimizu J. Yabuki T. Seyama Y. Hashimoto N. Biochem. Biophys. Res. Commun. 2000; 279: 62-68Crossref PubMed Scopus (22) Google Scholar). These observations, along with those of Min and Benzer (6Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar), suggest a potential role for BG1 in the biochemical pathology of X-linked adrenoleukodystrophy (XALD). XALD is a severe, often fatal, neurodegenerative disorder characterized by elevated plasma and tissue levels of saturated VLCFA (9Moser H.W. Smith K.D. Watkins P.A. Powers J. Moser A.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3257-3301Google Scholar). Organs primarily affected in the childhood cerebral form of XALD are brain, adrenal glands, and testes (9Moser H.W. Smith K.D. Watkins P.A. Powers J. Moser A.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3257-3301Google Scholar). Furthermore, decreased peroxisomal VLACS activity has been implicated in the biochemical pathology of this disorder (10Wanders R.J. van Roermund C.W. van Wijland M.J. Schutgens R.B. van den Bosch H. Schram A.W. Tager J.M. Biochem. Biophys. Res. Commun. 1988; 153: 618-624Crossref PubMed Scopus (135) Google Scholar, 11Lazo O. Contreras M. Bhushan A. Stanley W. Singh I. Arch. Biochem. Biophys. 1989; 270: 722-728Crossref PubMed Scopus (63) Google Scholar).Although these observations were suggestive of a link between BG1 and XALD, we were unable to establish a peroxisomal subcellular location for hBG1 when expressed in COS-1 cells (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Furthermore, we were unable to demonstrate any changes in VLCFA degradation in COS-1 cells overexpressing the protein (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Therefore, we sought to identify specific cell types that endogenously express BG1 in order to investigate the role of this enzyme in cellular fatty acid metabolism and to determine whether BG1 plays a role in the biochemical pathology of XALD.EXPERIMENTAL PROCEDURESMaterials and General Methods—[1-14C]Palmitic acid (C16:0), [1-14C]arachidonic acid (C20:4), [1-14C]docosahexaenoic acid (C22:6), and [1-14C]lignoceric acid (C24:0) were obtained from Moravek, Inc. [1-14C]Myristic acid (C14:0), [1-14C]stearic acid (C18:0), [1-14C]oleic acid (C18:1), and [1-14C]linoleic acid (C18:2) were obtained from American Radiolabeled Chemicals. Unlabeled fatty acids were from either Sigma or Cayman Chemicals. Protein was measured by the method of Lowry et al. (12Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). COS-1 cells, U373 glioma cells, rat astrocytes, and Neuro2a cells were maintained at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with penicillin/streptomycin and 10% fetal bovine serum (Invitrogen) in a 5% CO2 atmosphere. MA-10 cells were maintained in Weymouth's medium containing 15% horse serum, and Y-1 cells were cultured in minimal essential medium (Invitrogen) containing 10% fetal bovine serum. DNA sequencing was performed at the Johns Hopkins University, Department of Biological Chemistry Biosynthesis and Sequencing Facility using the fluorescent dideoxy terminator method of cycle sequencing on an Applied Biosystems Inc. 377 automated DNA sequencer following ABI protocols. General conditions for PCR were as reported previously (13Steinberg S.J. Wang S.J. Kim D.G. Mihalik S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1999; 257: 615-621Crossref PubMed Scopus (117) Google Scholar). COS-1 cells were transfected by electroporation as described previously (13Steinberg S.J. Wang S.J. Kim D.G. Mihalik S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1999; 257: 615-621Crossref PubMed Scopus (117) Google Scholar). Confocal microscopy was performed at the Microscope Facility, Johns Hopkins University School of Medicine. Statistical significance was calculated using Student's t test.cDNA Cloning and Expression—Full-length cDNA encoding hBG1 (GenBank™ accession number AF179481) was cloned into the mammalian expression vector pcDNA3 (Invitrogen) as described previously (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To obtain full-length cDNA encoding mBG1 (GenBank™ accession number NM_053178), we used the nucleotide sequence of hBG1 to search the mouse-expressed sequence tag data base and identified a single clone (GenBank™ accession number AA111606) that contained a full-length cDNA insert. The predicted amino acid sequence of this clone had 87% identity to hBG1. A 2740-bp insert containing 20-bp 3′-untranslated region, the 2115-bp open reading frame, and 545 bp of 5′-untranslated region was transferred to pcDNA3 after digestion with EcoRI and NotI, and the clone was resequenced.For purification of the hBG1 C-terminal polyclonal antibody, a maltose-binding protein/hBG1 (MBP-hBG1) fusion construct was prepared by excising a 1472-bp EcoRI/XbaI fragment from the hBG1/pcDNA3 clone and transferring it to pMalC2 (New England Biolabs). The fusion construct, encoding the C-terminal 472 amino acids of hBG1, was then transferred to Escherichia coli strain XL1-Blue.Antibody Generation and Purification—A peptide corresponding to the C-terminal 15 amino acids of hBG1 was synthesized, conjugated to keyhole limpet hemocyanin, and then used to immunize rabbits. These services were performed by a commercial contractor (Sigma-Genosys). For affinity purification, MBP-hBG1 fusion protein was expressed in E. coli by induction with isopropyl-β-d-thiogalactopyranoside. The bacterial pellet was lysed by sonication, and proteins were solubilized using 1% Triton X-100. After centrifugation, MBP-hBG1 was found in the insoluble fraction. This insoluble fraction was subjected to preparative SDS-PAGE on a 10% gel to resolve MBP-hBG1 protein from other bacterial proteins. Proteins were blotted onto a nitrocellulose membrane and detected with Ponceau S, and a strip containing the MBG-hBG1 protein was cut from the membrane. Crude antiserum was diluted with phosphate-buffered saline and incubated with the MBP-hBG1-containing membrane for 1 h at room temperature. After washing, bound antibody was eluted with 0.1 m glycine, pH 2.5, and the eluate was immediately neutralized by the addition of Tris-HCl, pH 8.0. The buffer was exchanged back to phosphate-buffered saline using a Centricon 30 (Millipore).Animals and Their Care—Wild type 129SvEv mice were obtained from Taconic, Inc. (Germantown, NY) and housed in facilities of the Johns Hopkins University School of Medicine. Animals were housed under controlled conditions between 22 and 27 °C on a 12-h light/dark cycle with food and water ad libitum. Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national and international laws and policies (EEC Council Directive 86/609,OJ L 358, 1 DEC.12, 1987 and National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996), U. S. National Research Council).In situ Hybridization—The method of Giger et al. (14Giger R.J. Wolfer D.P. De Wit G.M. Verhaagen J. J. Comp. Neurol. 1996; 375: 378-392Crossref PubMed Scopus (168) Google Scholar) was used with modification as described previously (15Mihalik S.J. Steinberg S.J. Pei Z. Park J. Kim D.G. Heinzer A.K. Dacremont G. Wanders R.J. Cuebas D.A. Smith K.D. Watkins P.A. J. Biol. Chem. 2002; 277: 24771-24779Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Sense and antisense probes corresponding to full-length mBG1 cDNA were prepared and labeled with digoxigenin as described previously (15Mihalik S.J. Steinberg S.J. Pei Z. Park J. Kim D.G. Heinzer A.K. Dacremont G. Wanders R.J. Cuebas D.A. Smith K.D. Watkins P.A. J. Biol. Chem. 2002; 277: 24771-24779Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Indirect Immunofluorescence and Immunohistochemistry—For indirect immunofluorescence analysis, cells were fixed in 4% formaldehyde in phosphate-buffered saline and permeabilized with 1.0% Triton X-100 prior to incubation with primary and secondary antibodies as described previously (16Watkins P.A. Gould S.J. Smith M.A. Braiterman L.T. Wei H.-M. Kok F. Moser A.B. Moser H.W. Smith K.D. Am. J. Hum. Genet. 1995; 57: 292-301PubMed Google Scholar). For immunohistochemistry, tissues from 3-month-old mice were harvested, quickly frozen in liquid nitrogen, and stored at -80 °C. Tissue sections were cut using a cryostat and fixed with 4% paraformaldehyde as described previously (15Mihalik S.J. Steinberg S.J. Pei Z. Park J. Kim D.G. Heinzer A.K. Dacremont G. Wanders R.J. Cuebas D.A. Smith K.D. Watkins P.A. J. Biol. Chem. 2002; 277: 24771-24779Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Brain sections were 20-μm-thick. Sections of liver, adrenal gland, testis, and ovary were 5-8-μm-thick. After fixation, sections were incubated for 30 min with 0.6% H2O2 in methanol followed by 20 min with 5% normal goat serum. Sections were then incubated sequentially with Avidin D and biotin for 15 min each (Avidin/Biotin blocking kit, Vector Laboratories). Incubation with primary rabbit antibody (affinity-purified anti-peptide antibody, 1:100) was for 1 h at 37 °C or overnight at 4 °C. Peroxidase-based detection was done using a Vectastain ABC kit (Vector Laboratories). After counterstaining with hematoxylin-Harris stain for 30 s, sections were dehydrated and mounted with DPX-mounting solution (Fluka Biochemika).Acyl-CoA Synthetase and Fatty Acid β-Oxidation Assays—Acyl-CoA synthetase assays utilizing radiolabeled substrates and fatty acid β-oxidation studies of labeled palmitic and lignoceric acids were performed as described previously (17Watkins P.A. Howard A.E. Mihalik S.J. Biochim. Biophys. Acta. 1994; 1214: 288-294Crossref PubMed Scopus (57) Google Scholar, 18Watkins P.A. Ferrell Jr., E.V. Pedersen J.I. Hoefler G. Arch. Biochem. Biophys. 1991; 289: 329-336Crossref PubMed Scopus (75) Google Scholar). The fluorometric assay of acyl-CoA synthetase activity was a coupled assay in which the acyl-CoA product was subsequently oxidized by acyl-CoA oxidase. H2O2 generated in the latter reaction was quantitated using a modification of the method of Guilbault et al. (19Guilbault G.G. Brignac Jr., P.J. Juneau M. Anal. Chem. 1968; 40: 1256-1263Crossref PubMed Scopus (340) Google Scholar). Each reaction tube contained, in a total volume of 250 μl, 10 nmol of unlabeled fatty acid (solubilized with α-cyclodextrin (18Watkins P.A. Ferrell Jr., E.V. Pedersen J.I. Hoefler G. Arch. Biochem. Biophys. 1991; 289: 329-336Crossref PubMed Scopus (75) Google Scholar)), 40 mm Tris(Cl-), pH 7.5, 10 mm ATP, 5 mm MgCl2, 0.2 mm CoA, 0.1% Triton X-100, 0.5 units of acyl-CoA oxidase (Sigma), 7.4 units of horseradish peroxidase (Sigma), 0.8 mm homovanillic acid (Sigma), and 15 μg of COS-1 cell protein. After a 10-min incubation at 37 °C, reactions were stopped by the addition of 700 μl of 0.83 m Na2CO3, pH 10.7, containing 12.5 mm EDTA. Fluorescence was detected using an Eppendorf Model 1020 fluorometer. For quantitation of fluorescence, a standard curve was created by incubating known amounts of H2O2 for 10 min at 37 °C in reaction mixtures lacking fatty acids and cellular protein.Northern Blot and RNA Dot Blot Analysis—Total RNA was prepared using the Trizol reagent (Invitrogen) from either fresh mouse tissues or tissues that were harvested, quick frozen in liquid nitrogen, and stored at -80 °C for less than two months. Twenty nanograms of RNA was electrophoresed on a 1% agarose gel at 4 V/cm for 2.5 h and then transferred overnight to a Hybond-N+ membrane (Amersham Biosciences). A cDNA probe for detection of mBG1 mRNA was prepared by PCR amplification of a 450-bp fragment using 5′-AGAGTCTCCAAGTCACGGTC-3′ and 5′-GGTGTAGATGCCAGTGACAAT-3′ as forward and reverse primers, respectively, and mouse brain cDNA as template. For control, a 528-bp glyceraldehyde-3-phosphate dehydrogenase probe was similarly prepared using 5′-ACCACCATGGAGAAGGCTGG-3′ and 5′-CTCAGTGTAGCCCAGGATGC-3′ as forward and reverse primers, respectively. Conditions for probe labeling, hybridization, and detection were as described previously (20Steinberg S.J. Wang S.J. McGuinness M.C. Watkins P.A. Mol. Genet. Metab. 1999; 68: 32-42Crossref PubMed Scopus (45) Google Scholar). A human multiple tissue expression membrane containing 76 tissues was obtained from Clontech. A cDNA probe for detection of hBG1 mRNA was prepared by PCR amplification of a 1138-bp fragment using 5′-CACGATGGAGGAATTCATGG-3′ and 5′-CACTCTGACCAGACTGATA-3′ as forward and reverse primers, respectively, and hBG1 cDNA (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) as template.Subcellular Fractionation—Neuro2a cells and mouse tissues were fractionated essentially as described by de Duve et al. (21de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2551) Google Scholar). Three confluent 10-cm dishes of Neuro2a cells were harvested by gentle trypsinization, washed with phosphate-buffered saline, and resuspended in 1.3 ml of ice-cold homogenization buffer (0.25 m sucrose, 10 mm Tris (Cl-), pH 8.0, 1 mm EDTA, and protease inhibitor mixture (Roche Applied Science)) and disrupted on ice using three passes through a precision ball-bearing homogenizer (22Balch W.E. Rothman J.E. Arch. Biochem. Biophys. 1985; 240: 413-425Crossref PubMed Scopus (210) Google Scholar). Mouse tissue (brain or liver) was homogenized in 5 volumes of the same buffer with a motor-driven Potter-Elvehjem homogenizer (3 passes). Homogenates were centrifuged (4 °C) for 30 s at 1500 × g to pellet nuclei (N). The post-nuclear supernatant was centrifuged for 15 min at 3500 × g to sediment a crude mitochondrial fraction (M). The post-mitochondrial supernatant was centrifuged for 10 min at 18,000 × g to obtain the peroxisome-enriched “light mitochondrial” fraction (L). The resulting supernatant was then centrifuged for 15 min at 435,000 × g to obtain a microsomal pellet (P) and a cytosolic supernatant (S). In some experiments, the crude mitochondrial fraction was further separated into a purified mitochondrial fraction and the mitochondria-associated membrane (MAM) fraction by centrifugation through a Percoll gradient as described by Vance and co-workers (23Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar, 24Stone S.J. Vance J.E. J. Biol. Chem. 2000; 275: 34534-34540Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar).RNA Interference—Three pairs of cDNA oligonucleotides containing complementary sense and antisense mBG1 sequences were obtained from Integrated DNA Technologies. Each 29-mer oligonucleotide contained an upstream AA dinucleotide, 19 bp of mBG1 sequence, and a downstream T7 promoter sequence (CCTGTCTC). Targeted sequences corresponded to bp 19-37, 633-651, and 1766-1784 of the mBG1-coding region. Small interfering RNA (siRNA) 21-mers were synthesized using the Silencer kit (Ambion) following the manufacturer's instructions. As a control, siRNA corresponding to bp 4-24 of the mouse serum albumin (mSA) coding region was also synthesized. Neuro2a cells (in 100-mm culture dishes, ∼35% confluent) were transfected with a mixture of the three mBG1-specific siRNAs or the mSA-specific siRNA using the siPORT Amine reagent (Ambion). 30 μl of siPORT amine was mixed with 560 μl of Opti-MEM I (Invitrogen) and incubated for 15 min at room temperature. siRNAs were added to a final concentration of 50 nm, and the incubation was continued for 10 min. This mixture was then added at a final concentration of 5 nm to cells in 2.4 ml of Opti-MEM I. After incubation for 16 h at 37 °C in the cell culture incubator, 7 ml of usual culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum) was added and incubation at 37 °C was continued for up to 6 days.RESULTSSubstrate Specificity of hBG1 Expressed in COS-1 Cells— COS-1 cells were transiently transfected with hBG1 cDNA, and the increase in cellular ACS activity due to expression of hBG1 protein was measured. We previously demonstrated that hBG1 had both LACS and VLACS activity using, respectively, radiolabeled palmitic acid (C16:0) and lignoceric acid (C24:0) substrates (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To extend our knowledge of the substrate specificity of this enzyme, we examined the ACS activity toward several saturated, monounsaturated, and polyunsaturated fatty acids using a coupled fluorometric assay. Compared with vector-transfected control COS-1 cells, hBG1-expressing cells activated several saturated fatty acids (C14:0, C16:0, and C18:0), the ω9 monounsaturated fatty acids oleic (C18:1), erucic (C22: 1), and nervonic (C24:1), the ω6 polyunsaturated fatty acids linoleic (C18:2) and arachidonic (C20:4), and the ω3 polyunsaturated fatty acids α-linoleic (C18:3), eicosapentaenoic (C20:5), and docosahexaenoic (C22:6) (Fig. 1). In contrast, hBG1 failed to activate either the β-methyl branched-chain fatty acid phytanic acid or the α-methyl branched-chain fatty acid pristanic acid (data not shown). Thus, when overexpressed in COS-1 cells, hBG1 appears to have a diverse substrate specificity and is able to activate a broad spectrum of unbranched fatty acids. The fluorometric assay was not sensitive enough to detect activation of lignoceric acid (data not shown).Identification of Cell Types Endogenously Expressing mBG1—Previously, using a human multiple tissue Northern blot containing only eight different tissues, we reported that hBG1 was expressed primarily in brain (5Steinberg S.J. Morgenthaler J. Heinzer A.K. Smith K.D. Watkins P.A. J. Biol. Chem. 2000; 275: 35162-35169Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Subsequently, Moriya-Sato et al. (7Moriya-Sato A. Hida A. Inagawa-Ogashiwa M. Wada M.R. Sugiyama K. Shimizu J. Yabuki T. Seyama Y. Hashimoto N. Biochem. Biophys. Res. Commun. 2000; 279: 62-68Crossref PubMed Scopus (22) Google Scholar) reported that in the mouse, BG1 was expressed in adrenal gland and testis as well and Tang et al. (8Tang P.Z. Tsai-Morris C.H. Dufau M.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6581-6586Crossref PubMed Scopus (25) Google Scholar) noted that rat BG1 was also expressed in ovary. A Northern blot prepared using total RNA from tissues of 3-month-old 129SvEv mice and probed with an mBG1-specific probe indicated that mBG1 was expressed in brain, adrenal gland, testis, ovary, and spleen (Fig. 2A). An RNA dot blot representing 76 different human tissues was also examined with an hBG1-specific antisense probe (Fig. 2B). All of the regions of human brain with the exception of pituitary gland highly expressed hBG1. Modest expression was detected in human testis and adrenal gland, but hBG1 message was not very abundant in ovary or spleenFig. 2Expression of BG1 in mouse and human tissues.A, a Northern blot of mouse tissues was prepared and hybridized with an mBG1-specific probe as described under “Experimental Procedures.” The mBG1 transcript (upper panel) was ∼3.3 kb in all of the tissues in which it was expressed. The blot was also hybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe (lower panel) as a loading control. B, a human Multiple Tissue Expression (Clontech) RNA dot blot was probed with an hBG1-specific probe. All of the regions of the brain (columns A, B, and C) with the exception of pituitary gland (C4) had the strongest signals. Brain regions included whole brain, cerebral cortex, frontal lobe, parietal lobe, occipital lobe, temporal lobe, precentral gyrus, pons (column A, rows 1-8, respectively), left cerebellum, right cerebellum, corpus callosum, amygdala, caudate nucleus, hippocampus, medulla oblongata, putamen (column B, rows 1-8, respectively), substantia nigra, accumbens nucleus, thalamus (column C, rows 1-3, respectively), and spinal cord (C5). C6, C7, and C8 contain no R
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