The Second Member of the Human and Murine “Bubblegum” Family Is a Testis- and Brainstem-specific Acyl-CoA Synthetase
2005; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m511558200
ISSN1083-351X
AutoresZhengtong Pei, Zhenzhen Jia, Paul A. Watkins,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoAcyl-CoA synthetases that activate fatty acids to their CoA derivatives play a central role in fatty acid metabolism. ACSBG1, an acyl-CoA synthetase originally identified in the fruit fly mutant bubblegum, was hypothesized to contribute to the biochemical pathology of X-linked adrenoleukodystrophy. We looked for homologous proteins and identified ACSBG2 in humans, mice, and rats. Human ACSBG1 and ACSBG2 amino acid sequences are 50% identical. ACSBG2 expression was confined to the testis and brainstem. Immunohistochemistry and in situ hybridization studies further localized ACSBG2 expression to testicular Sertoli cells and large motoneurons in the medulla oblongata and cervical spinal cord. Full-length cDNA encoding human and mouse ACSBG2 was cloned. In transfected COS-1 cells, both human and murine ACSBG2 were detected as 75- to 80-kDa proteins by Western blot. Cells overexpressing ACSBG2 had increased ability to activate oleic acid (C18:1ω9) and linoleic acid (C18:2ω6) but not other fatty acid substrates tested. Within a highly conserved motif known to be important for catalysis, human ACSBG2 contains a histidine residue where all other known acyl-CoA synthetases, including mouse and rat ACSBG2, contain an arginine. This substitution resulted in a shift of the human ACSBG2 pH optimum to a more acidic pH. Mutation of this histidine to arginine improved catalytic function at neutral pH by shifting the pH profile without affecting substrate specificity. Although the role of ACSBG2 in testicular and neuronal lipid metabolism remains unclear, the limited tissue expression pattern and limited substrate specificity rule out a likely role for this enzyme in X-linked adrenoleukodystrophy pathology. Acyl-CoA synthetases that activate fatty acids to their CoA derivatives play a central role in fatty acid metabolism. ACSBG1, an acyl-CoA synthetase originally identified in the fruit fly mutant bubblegum, was hypothesized to contribute to the biochemical pathology of X-linked adrenoleukodystrophy. We looked for homologous proteins and identified ACSBG2 in humans, mice, and rats. Human ACSBG1 and ACSBG2 amino acid sequences are 50% identical. ACSBG2 expression was confined to the testis and brainstem. Immunohistochemistry and in situ hybridization studies further localized ACSBG2 expression to testicular Sertoli cells and large motoneurons in the medulla oblongata and cervical spinal cord. Full-length cDNA encoding human and mouse ACSBG2 was cloned. In transfected COS-1 cells, both human and murine ACSBG2 were detected as 75- to 80-kDa proteins by Western blot. Cells overexpressing ACSBG2 had increased ability to activate oleic acid (C18:1ω9) and linoleic acid (C18:2ω6) but not other fatty acid substrates tested. Within a highly conserved motif known to be important for catalysis, human ACSBG2 contains a histidine residue where all other known acyl-CoA synthetases, including mouse and rat ACSBG2, contain an arginine. This substitution resulted in a shift of the human ACSBG2 pH optimum to a more acidic pH. Mutation of this histidine to arginine improved catalytic function at neutral pH by shifting the pH profile without affecting substrate specificity. Although the role of ACSBG2 in testicular and neuronal lipid metabolism remains unclear, the limited tissue expression pattern and limited substrate specificity rule out a likely role for this enzyme in X-linked adrenoleukodystrophy pathology. Acyl-CoA synthetases (ACS) 2The abbreviations used are: ACS, acyl-CoA synthetase; h, human; m, mouse; r, rat; X-ALD, X-linked adrenoleukodystrophy; PBS, phosphate-buffered saline; EST, expressed sequence tag; Triacsin C, 1-hydroxy-3-(E,E,E-2′,4′,7′-undecatrienylidine)-triazene.2The abbreviations used are: ACS, acyl-CoA synthetase; h, human; m, mouse; r, rat; X-ALD, X-linked adrenoleukodystrophy; PBS, phosphate-buffered saline; EST, expressed sequence tag; Triacsin C, 1-hydroxy-3-(E,E,E-2′,4′,7′-undecatrienylidine)-triazene. carry out a fundamental reaction in fatty acid metabolism, thioesterification of the acyl group to coenzyme A (CoA) (1Watkins P.A. Prog. Lipid Res. 1997; 36: 55-83Crossref PubMed Scopus (154) Google Scholar). The fatty acyl-CoA product can have several metabolic fates, including incorporation into phospholipids, mono-, di-, and triacylglycerols, sphingolipids, glycolipids, or cholesterol esters, acylation of proteins, and catabolism via β-oxidation. All ACSs are thought to utilize the same ATP-dependent reaction mechanism, in which the fatty acid is first adenylated with release of inorganic pyrophosphate. In the second step of the reaction, CoA-SH displaces AMP by forming a thioester linkage with the fatty acyl group (1Watkins P.A. Prog. Lipid Res. 1997; 36: 55-83Crossref PubMed Scopus (154) Google Scholar). Thus, all ACSs are thought to contain a highly conserved amino acid sequence referred to at the AMP-binding domain (PROSITE PDOC00427, available at au.expasy.org/cgi-bin/nicedoc.pl?PDOC00427). We (2Steinberg 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 (105) Google Scholar) and others (3Black 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) identified a second highly conserved region in the amino acid sequences of known ACSs. Bioinformatics approaches, using amino acid and nucleotide sequences derived from the various genome sequencing projects, have allowed us to estimate that mammals, including humans, have at least 25 distinct enzymes that are documented or putative ACSs. 3P. A. Watkins, submitted for publication.3P. A. Watkins, submitted for publication. In the Drosophila mutant "bubblegum" (4Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar), the defective gene product, ACSBG1, 4A uniform nomenclature for long-chain ACSs was approved by the Nomenclature Commission of the Human Genome Organization (35Mashek D.G. Bornfeldt K.E. Coleman R.A. Berger J. Bernlohr D.A. Black P. DiRusso C.C. Farber S.A. Guo W. Hashimoto N. Khodiyar V. Kuypers F.A. Maltais L.J. Nebert D.W. Renieri A. Schaffer J.E. Stahl A. Watkins P.A. Vasiliou V. Yamamoto T.T. J. Lipid Res. 2004; 45: 1958-1961Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and adaptation of this system for several other ACS families, including the "bubblegum" family, was recently approved. Approved gene names ACSBG1 and ACSBG2 are used to designate genes formerly called BG1 and BGR, respectively.4A uniform nomenclature for long-chain ACSs was approved by the Nomenclature Commission of the Human Genome Organization (35Mashek D.G. Bornfeldt K.E. Coleman R.A. Berger J. Bernlohr D.A. Black P. DiRusso C.C. Farber S.A. Guo W. Hashimoto N. Khodiyar V. Kuypers F.A. Maltais L.J. Nebert D.W. Renieri A. Schaffer J.E. Stahl A. Watkins P.A. Vasiliou V. Yamamoto T.T. J. Lipid Res. 2004; 45: 1958-1961Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and adaptation of this system for several other ACS families, including the "bubblegum" family, was recently approved. Approved gene names ACSBG1 and ACSBG2 are used to designate genes formerly called BG1 and BGR, respectively., 5GenBank™ accession numbers: hACSBG2, NM_030924; mACSBG2, DQ250679; and rACSBG2, XM_236792.5GenBank™ accession numbers: hACSBG2, NM_030924; mACSBG2, DQ250679; and rACSBG2, XM_236792. was found to be an ACS (2Steinberg 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 (105) Google Scholar, 5Pei Z. Oey N.A. Zuidervaart M.M. Jia Z. Li Y. Steinberg S.J. Smith K.D. Watkins P.A. J. Biol. Chem. 2003; 278: 47070-47078Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Several properties of ACSBG1 suggest that it may be involved in the biochemical pathology of X-linked adrenoleukodystrophy (X-ALD). Patients with X-ALD (6Moser 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. McGraw-Hill, New York2001: 3257-3301Google Scholar), as well as male fruit flies lacking ACSBG1 (4Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar), have neurodegeneration and elevated tissues levels of saturated very long-chain fatty acids. In mice, ACSBG1 is highly expressed in the brain (hippocampal and hypothalamic neurons and cerebellar Purkinje cells), adrenal cortex (zona fasciculata), and testis (Leydig cells) (5Pei Z. Oey N.A. Zuidervaart M.M. Jia Z. Li Y. Steinberg S.J. Smith K.D. Watkins P.A. J. Biol. Chem. 2003; 278: 47070-47078Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These tissues are the primary sites of pathology in X-ALD (2Steinberg 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 (105) Google Scholar, 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 (23) Google Scholar). When expressed in COS-1 cells, ACSBG1 has robust acyl-CoA synthetase activity and is capable of activating long- and very long-chain saturated, monounsaturated, and polyunsaturated fatty acids (2Steinberg 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 (105) Google Scholar, 5Pei Z. Oey N.A. Zuidervaart M.M. Jia Z. Li Y. Steinberg S.J. Smith K.D. Watkins P.A. J. Biol. Chem. 2003; 278: 47070-47078Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). However, a direct role for ACSBG1 in X-ALD pathology has not yet been demonstrated (8Fraisl P. Forss-Petter S. Zigman M. Berger J. Biochem. J. 2004; 377: 85-93Crossref PubMed Scopus (25) Google Scholar, 9Jia Z. Pei Z. Li Y. Wei L. Smith K.D. Watkins P.A. Mol. Genet. Metab. 2004; 83: 117-127Crossref PubMed Scopus (22) Google Scholar). Therefore, it was of interest to determine whether ACS enzymes related to ACSBG1 existed, and if so, whether they were involved in X-ALD biochemical pathology. Examination of the ACSBG1 amino acid sequence revealed that it contained both the AMP-binding domain and the second conserved ACS sequence (motif 2) described above (2Steinberg 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 (105) Google Scholar). Sequence homology within motif 2 can be used to group related proteins into families 3P. A. Watkins, submitted for publication. (2Steinberg 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 (105) Google Scholar). However, the ACSBG1 motif 2 sequence differed sufficiently from motif 2 sequences of known short-, medium-, long-, and very long-chain ACSs, thus we concluded that ACSBG1 belonged to a distinct ACS family (2Steinberg 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 (105) Google Scholar). Homology probing of the National Center for Biotechnology Information (NCBI) protein and nucleotide databases revealed that the ACS gene/protein family of humans, mice, and fruit flies to which ACSBG1 belongs each has only one other member. This "bubblegum-related" gene (ACSBG2) encodes a protein that is an ACS with a very restricted tissue and cellular expression pattern. Furthermore, the native human protein has a naturally occurring mutation at a highly conserved position that affects its enzymatic function. We describe here some of the properties of human and mouse ACSBG2. Materials and General Methods—[1-14C]Lauric acid (C12:0), [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]Oleic acid (C18:1) and [1-14C]linoleic acid (C18:2) were obtained from American Radiolabeled Chemicals. [1-14C]Stearic acid was obtained from Amersham Biosciences. Unlabeled fatty acids were from either Sigma or Cayman Chemical. Protein was measured by the method of Lowry et al. (10Lowry 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 (American Type Culture Collection) were grown 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. TM-4 Sertoli cells were obtained from American Type Culture Collection and grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 10% fetal bovine serum. General conditions for PCR were as previously reported (11Steinberg S.J. Wang S.J. Kim D.G. Mihalik S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1999; 257: 615-621Crossref PubMed Scopus (118) Google Scholar). DNA sequencing was performed at the Johns Hopkins University Department of Biological Chemistry Biosynthesis and Sequencing Facility using the fluorescent di-deoxy terminator method of cycle sequencing on an Applied Biosystems Inc. 377 automated DNA sequencer, following the protocols of the manufacturer. COS-1 cells were transfected by electroporation as described previously (11Steinberg S.J. Wang S.J. Kim D.G. Mihalik S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1999; 257: 615-621Crossref PubMed Scopus (118) Google Scholar). TM4 cells were transfected using Oligofectamine (Invitrogen). Statistical significance was calculated using Student's t test. Animals and Their Care—Wild-type 129SvEv mice were obtained from Taconic, Inc. (Germantown, NY). All mice used in these studies were approximately 3 months of age. Mice were housed in facilities of the Johns Hopkins University School of Medicine under controlled conditions, between 22 °C 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; NIH Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). Cloning of Full-length ACSBG2 cDNA—Human testis total RNA (Ambion) was reverse-transcribed using the ThermoScript™ reverse transcription-PCR First Strand cDNA Synthesis kit (Invitrogen) to obtain testis cDNA. Full-length ACSBG2 cDNA was obtained by PCR amplification using the forward oligonucleotide primer 5′-CGCGAATTCCTGCACACCTGGAATGAC-3′ (P15-1), which incorporates an EcoRI restriction site, reverse primer 5′-ATAGCGGCCGCCAGTCAGTGGTACATGTG-3′ (P15-6), which incorporates a NotI site, and testis cDNA as template. The 2016-bp PCR product was cloned into the EcoRI and NotI sites of the mammalian expression vector pcDNA3 (Invitrogen). The resulting construct, which included the entire open reading frame and 13 bp of 5′-untranslated DNA but no additional 3′ sequence following the stop codon, was fully sequenced. The insert sequence was identical to that of GenBank™ accession number NM_030924. Mouse ACSBG2 cDNA was cloned using a similar strategy. Total RNA was prepared from fresh mouse testis using the TRIzol reagent (Invitrogen). cDNA was prepared by reverse transcription and used as template for PCR with forward primer 5′-GAATTCAAAGGCTGGACCACCAATGACTC-3′, which incorporates an EcoRI site, and reverse primer 5′-CTCGAGGCTGATCTGTTGGAGGCATGAGGA-3′, which incorporates an XhoI site. The purified PCR product was cloned into the EcoRI and XhoI sites of pcDNA3. The insert was sequenced completely and contained a complete open reading frame, encoding 667 amino acids, plus 16 bp of 5′-untranslated DNA and 62 bp of 3′-untranslated DNA. The sequence of the amino-terminal portion (up to the codon for amino acid 243 of the open reading frame) was identical to that of GenBank™ accession number XM_619478, whereas the sequence of the C-terminal portion (encoding residue 244 through the 3′-untranslated region) was identical to that of XM_286829. The complete nucleotide sequence has been submitted to GenBank™ (accession number DQ250679). Polyclonal Antibody Production and Purification—A 951-bp fragment containing the C-terminal 315 amino acids of hACSBG2 was amplified by PCR using forward primer 5′-GGATCCGCAACATTGGCTTCAAGGTCAA-3′, incorporating a BamHI site, reverse primer P15-6, and full-length hACSBG2 in pcDNA3 as template. The PCR product was digested with BamHI and NotI and cloned into the bacterial expression vector pGEX5X2 (Amersham Biosciences). After transduction of Escherichia coli BL21DE3 cells, synthesis of the glutathione S-transferase-hACSBG2 fusion protein was induced by incubation with 1 mm isopropyl-β-d-thiogalactopyranoside. The fusion protein was solubilized from sonicated bacterial cells using 1% Triton X-100 and purified by chromatography on glutathione-Sepharose (Amersham Biosciences). Immunization of rabbits with purified fusion protein, boosting and bleeding was done commercially (Cocalico Biologicals, Reamstown, PA). Crude antiserum was purified by either affinity binding or by adsorption. For affinity purification, preparative SDS-PAGE of the fusion protein on a 10% gel was used. After loading fusion protein in sample buffer and electrophoresis for 5 min, the power was turned off and a second aliquot of sample was loaded. This process was repeated such that a total of six aliquots were present, after which electrophoresis was allowed to proceed to completion. Proteins were transferred to nitrocellulose and identified by Ponceau-S staining. After washing with phosphate-buffered saline (PBS), the nitrocellulose strip containing the six bands of fusion protein was incubated with a 5-fold dilution of crude serum (in PBS) overnight at 4 °C. The membrane was washed with PBS, bound antibodies were eluted with 0.1 m glycine, pH 2.5, and the eluate was immediately neutralized by addition of 1 m Tris-HCl, pH 8.0. The buffer was exchanged back to PBS using a Centricon 30 (Millipore), 1% bovine serum albumin was added, and purified antibody was stored at -80 °C. For adsorption of the crude antiserum, a nitrocellulose membrane was prepared by electrophoretic transfer of a mixture of mouse liver proteins and COS-1 cell proteins, separated by preparative SDS-PAGE. Because neither liver nor kidney (origin of COS-1 cells) express ACSBG2, dilute antiserum was incubated with this membrane to adsorb nonspecific antibodies. Adsorbed antiserum was stored at -80 °C. Site-directed Mutagenesis—The overlap extension method was used to mutate histidine 511 of hACSBG2 to arginine (12Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6825) Google Scholar). PCR was used to amplify two fragments of hACSBG2 that overlap and that each incorporate the desired mutation (underlined in the oligonucleotide primers); both reactions used full-length hACSBG2 in pcDNA3 as template. The first reaction amplified a 1547-bp fragment encoding amino acids 1-511 using forward primer P15-1 and reverse primer 5′-TGCGGCCGGTGACATAGAGGA-3′. The second reaction amplified a 482-bp fragment that encodes amino acids 508-666 using forward primer 5′-GTCACCGGCCGCATCAAAGAA-3 and reverse primer P15-6. The underlined CGC (R) codon replaced the wild-type CAC (H) codon. The two PCR products were gel-purified and used as template for additional PCR, with forward oligonucleotide primer P15-1 and reverse primer P15-6. The 2016-bp product was cloned into pcDNA3 as described above and completely re-sequenced. The sequence matched that of NM_030924 completely, except for the engineered A→G mutation. Northern Blot and mRNA Dot Blot Analyses—Total RNA was prepared from freshly harvested mouse tissues using the TRIzol reagent (Invitrogen). Twenty μg 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 mAcsbg2 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 testis 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 previously described (13Steinberg S.J. Wang S.J. McGuinness M.C. Watkins P.A. Mol. Genet. Metab. 1999; 68: 32-42Crossref PubMed Scopus (46) Google Scholar). A human multiple tissue expression array containing 76 tissues was obtained from Clontech. A cDNA probe for detection of hACSBG2 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 hACSBG2 cDNA as template. Subcellular Fractionation and Western Blotting—Mouse tissues were homogenized in 5 volumes of buffer (0.25 m sucrose, 10 mm Tris-Cl, pH 8.0, 1 mm EDTA) containing protease inhibitor mixture (Complete, Roche Applied Science) using a Pellet Pestle homogenizer (Kimble/Kontes). Subcellular fractions were prepared essentially following the method of de Duve et al. (14de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2565) Google Scholar) as previously described (5Pei Z. Oey N.A. Zuidervaart M.M. Jia Z. Li Y. Steinberg S.J. Smith K.D. Watkins P.A. J. Biol. Chem. 2003; 278: 47070-47078Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). For Western blots, cells were harvested by gentle trypsinization, washed with PBS containing protease inhibitor mixture, and solubilized in Laemmli sample buffer (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar). Proteins were separated by SDS-PAGE on 10% gels, and transferred to nitrocellulose membranes; after incubation with primary antibody and horseradish peroxidase-conjugated secondary antibody, immunoreactive proteins were detected using the SuperSignal West Pico chemiluminescent reagent (Pierce). Acyl-CoA Synthetase Assays—Radiochemical assays of ACS activity in frozen/thawed COS-1 cell suspensions using [1-14C]fatty acid substrates were performed essentially as described (16Watkins P.A. Ferrell Jr., E.V. Pedersen J.I. Hoefler G. Arch. Biochem. Biophys. 1991; 289: 329-336Crossref PubMed Scopus (75) Google Scholar, 17Watkins P.A. Howard A.E. Mihalik S.J. Biochim. Biophys. Acta. 1994; 1214: 288-294Crossref PubMed Scopus (57) Google Scholar). COS-1 cells, 3 days post-transfection with either ACSBG2 constructs or the empty pcDNA3 vector, were harvested by gentle trypsinization, washed with phosphate-buffered saline, and resuspended in 0.25 m sucrose containing 10 mm Tris (Cl-), pH 8.0, 1 mm EDTA, and protease inhibitor mixture (Complete, Roche Applied Science). Cell suspensions were subjected to at least one freeze/thaw cycle (-80 °C) prior to assay. In some cases, as noted in the text and figure legends, either KCl was added to the reaction mixture, or potassium phosphate buffer, pH 7.5, replaced Tris (Cl-), pH 7.5. When the inhibitor Triacsin C was used, the assay was modified to permit preincubation of enzyme with the inhibitor prior to adding the radiolabeled substrate. Triacsin C (final concentration, 10 μm) in ethanol was added to reaction mixes containing all components except 14C-fatty acid. An equal volume of ethanol (final concentration, 1%) was added to control incubations. After 15 min at 37 °C, labeled substrate solubilized in α-cyclodextrin (16Watkins P.A. Ferrell Jr., E.V. Pedersen J.I. Hoefler G. Arch. Biochem. Biophys. 1991; 289: 329-336Crossref PubMed Scopus (75) Google Scholar) was added and the reaction allowed to proceed for 20 min as usual. Fluorometric ACS assays were performed as previously described (5Pei Z. Oey N.A. Zuidervaart M.M. Jia Z. Li Y. Steinberg S.J. Smith K.D. Watkins P.A. J. Biol. Chem. 2003; 278: 47070-47078Abstract Full Text Full Text PDF PubMed Scopus (59) 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 (18Watkins 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, testis, brainstem, and spinal cord from 3-month-old mice were harvested, quickly frozen in liquid nitrogen, and stored at -80 °C. Tissue sections (5-8 μm thick) were cut using a cryostat and fixed with 4% paraformaldehyde; blocking, incubation with primary and secondary antibody, peroxidase-based detection, counterstaining, and mounting were performed as described previously (5Pei Z. Oey N.A. Zuidervaart M.M. Jia Z. Li Y. Steinberg S.J. Smith K.D. Watkins P.A. J. Biol. Chem. 2003; 278: 47070-47078Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In Situ Hybridization—The method of Giger et al. (19Giger 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 previously described (20Mihalik 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 (87) Google Scholar). Sense and antisense probes corresponding to full-length hACSBG2 cDNA were prepared and labeled with digoxigenin as previously described (20Mihalik 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 (87) Google Scholar). Identification and Predicted Characteristics of ACSBG2—The ACSBG1 gene encodes a protein found in several species, including fruit flies (4Min K.T. Benzer S. Science. 1999; 284: 1985-1988Crossref PubMed Scopus (145) Google Scholar), mice (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 (23) Google Scholar), rats (21Tang P.Z. Tsai-Morris C.H. Dufau M.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6581-6586Crossref PubMed Scopus (26) Google Scholar), and humans (2Steinberg 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 (105) Google Scholar). Transfected cells expressing human, mouse, or rat ACSBG1 cDNA exhibited increased ACS activity (2Steinberg 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 (105) Google Scholar, 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 (23) Google Scholar, 21Tang P.Z. Tsai-Morris C.H. Dufau M.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6581-6586Crossref PubMed Scopus (26) Google Scholar). To determine whether mammalian genomes contained related proteins, we queried NCBI nucleotide and protein databases using the BLAST algorithm with the human (h) ACSBG1 sequence as probe. We identified one predicted human protein consisting of 666 amino acid residues that shared 50% identity and 69% similarity with hACSBG1 (Fig. 1). Two highly conserved amino acid motifs (solid and broken underlines in Fig. 1) characteristic of ACSs were present in this open reading frame, hereafter referred to as hACSBG2. Within motif 2 (broken underline), the sequence that facilitates segregation of ACSs into families of related proteins, hACSBG1 and the predicted protein share 68% identity and 89% similarity. The next most homologous human proteins identified in the BLAST search had <30% identity with hACSBG1 and were members of the long-chain ACS family. The gene encoding hACSBG2 had previously been referred to as "bubblegum-related" with the interim gene symbol BGR. The BLAST search also detected putative mouse (m) and rat (r) homologs of hACSBG2, each of which contains 667 amino acids (Fig. 2).FIGURE 2Alignment of human, mouse, and rat ACSBG2 amino acid sequences. The predicted amino acid sequences of hACSBG2, mACSBG2, and rACSBG2 were aligned using ClustalW. The AMP-binding domain (solid line) and conserved motif 2 (dashed line) are indicated. Mouse and rat ACSBG2 preserve the invariant arginine residue (asterisk) that was found to be a histidine residue in human ACSBG2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Human ACSBG2 has a theoretical molecular weight of 74,413 Da and a pI of 8.59 (us.expasy.org/tools/pi_tool.html). No signal peptide sequence, mitochondrial targeting signal, or peroxisomal targeting signals were evident. Potential N-glycosylation sites were detected at residues 19 and 246 by the NetNGlyc 1.0 program (available at www.cbs.dtu.dk/services/NetNGlyc/). No potential O-glycosylation sites were found. 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