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• W2006966998 abstract "Here we report the identification of a previously uncharacterized human protein as the human monolysocardiolipin acyltransferase-1 (MLCL AT-1). Pig liver mitochondria were treated with n-butyl alcohol followed by Q-Sepharose chromatography, preparative gel electrophoresis, cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose chromatography, and finally monolysocardiolipin-adriamycin-agarose affinity chromatography. Elution with either monolysocardiolipin or linoleoyl coenzyme A revealed a major band at 74 kDa with high specific activity (2,300 pmol/min/mg) for the acylation of monolysocardiolipin to cardiolipin using [1-14C]linoleoyl coenzyme A as substrate. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis followed by search of the Mascot protein data base revealed peptide matches consistent with a 59-kDa protein identified as unknown human protein (GenBankTM protein accession number AAX93141; nucleotide accession number AC011742.3). The purified human recombinant MLCL AT-1 protein utilized linoleoyl coenzyme A > oleoyl coenzyme A > palmitoyl coenzyme A for the specific acylation of monolysocardiolipin to cardiolipin. Expression of MLCL AT-1 in HeLa cells increased mitochondrial monolysocardiolipin acyltransferase activity and [1-14C]linoleic acid incorporated into cardiolipin, whereas RNA interference knockdown of MLCL AT-1 in HeLa cells resulted in reduction in enzyme activity and [1-14C]linoleic acid incorporated into cardiolipin. In contrast, expression of MLCL AT-1 in HeLa cells did not alter [1-14C]oleic or [1-14C]palmitate incorporation into cardiolipin indicating in vivo specificity for the remodeling of cardiolipin with linoleate. Finally, expression of MLCL AT-1 in Barth syndrome lymphoblasts, which exhibit cardiolipin levels 20% that of normal lymphoblasts, increased mitochondrial monolysocardiolipin acyltransferase activity, [1-14C]linoleic acid incorporation into cardiolipin, cardiolipin mass, and succinate dehydrogenase (mitochondrial complex II) activity compared with mock-transfected Barth syndrome lymphoblasts. The results identify MLCL AT-1 as a human mitochondrial monolysocardiolipin acyltransferase involved in the remodeling of cardiolipin. Here we report the identification of a previously uncharacterized human protein as the human monolysocardiolipin acyltransferase-1 (MLCL AT-1). Pig liver mitochondria were treated with n-butyl alcohol followed by Q-Sepharose chromatography, preparative gel electrophoresis, cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose chromatography, and finally monolysocardiolipin-adriamycin-agarose affinity chromatography. Elution with either monolysocardiolipin or linoleoyl coenzyme A revealed a major band at 74 kDa with high specific activity (2,300 pmol/min/mg) for the acylation of monolysocardiolipin to cardiolipin using [1-14C]linoleoyl coenzyme A as substrate. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis followed by search of the Mascot protein data base revealed peptide matches consistent with a 59-kDa protein identified as unknown human protein (GenBankTM protein accession number AAX93141; nucleotide accession number AC011742.3). The purified human recombinant MLCL AT-1 protein utilized linoleoyl coenzyme A > oleoyl coenzyme A > palmitoyl coenzyme A for the specific acylation of monolysocardiolipin to cardiolipin. Expression of MLCL AT-1 in HeLa cells increased mitochondrial monolysocardiolipin acyltransferase activity and [1-14C]linoleic acid incorporated into cardiolipin, whereas RNA interference knockdown of MLCL AT-1 in HeLa cells resulted in reduction in enzyme activity and [1-14C]linoleic acid incorporated into cardiolipin. In contrast, expression of MLCL AT-1 in HeLa cells did not alter [1-14C]oleic or [1-14C]palmitate incorporation into cardiolipin indicating in vivo specificity for the remodeling of cardiolipin with linoleate. Finally, expression of MLCL AT-1 in Barth syndrome lymphoblasts, which exhibit cardiolipin levels 20% that of normal lymphoblasts, increased mitochondrial monolysocardiolipin acyltransferase activity, [1-14C]linoleic acid incorporation into cardiolipin, cardiolipin mass, and succinate dehydrogenase (mitochondrial complex II) activity compared with mock-transfected Barth syndrome lymphoblasts. The results identify MLCL AT-1 as a human mitochondrial monolysocardiolipin acyltransferase involved in the remodeling of cardiolipin. Cardiolipin (CL) 2The abbreviations used are: CLcardiolipinMLCLmonolysocardiolipinMLCL ATmonolysocardiolipin acyltransferaseTAZtafazzinMALDI-TOFmatrix activated laser desorption ionization-time of flightBTHSBarth syndromeALCAT1acyllysocardiolipin acyltransferase-1MLCL AT-1monolysocardiolipin acyltransferase-1ERendoplasmic reticulumRNAiRNA interference. is a major phospholipid found in mammalian mitochondria with a multitude of biological functions (reviewed in Refs. 1Hatch G.M. Biochem. Cell Biol. 2004; 82: 99-112Crossref PubMed Scopus (94) Google Scholar, 2Houtkooper R.H. Vaz F.M. Cell. Mol. Life Sci. 2008; 65: 2493-2506Crossref PubMed Scopus (304) Google Scholar, 3Schlame M. J. Lipid. Res. 2008; 49: 1607-1620Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 4Hostetler K.Y. Phospholipids.in: Hawthorne J.N. Ansell G.B. Elsevier Science Publishers B.V., Amsterdam1982: 215-261Google Scholar, 5Schlame M. Rua D. Greenberg M.L. Prog. Lipid Res. 2000; 39: 257-288Crossref PubMed Scopus (665) Google Scholar, 6Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (789) Google Scholar, 7Chicco A.J. Sparagna G.C. Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (498) Google Scholar). For example, CL is responsible for modulation of the activity of several mitochondrial enzymes involved in the generation of ATP (reviewed in Refs. 8Hoch F.L. Biochim. Biophys. Acta. 1992; 1113: 71-133Crossref PubMed Scopus (546) Google Scholar, 9Hatch G.M. Int. J. Mol. Med. 1998; 1: 33-41PubMed Google Scholar). In fact, it has been suggested that CL is the “glue” that holds the mitochondrial respiratory complex together (10Zhang M. Mileykovskaya E. Dowhan W. J. Biol. Chem. 2002; 277: 43553-43556Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). The role of CL in genetic diseases such as Barth syndrome, a rare X-linked genetic disorder, is beginning to emerge. Barth syndrome is the only known genetic disease in which the specific biochemical defect is a reduction in CL and accumulation of monolysocardiolipin (MLCL) caused by mutations in the TAZ gene (reviewed in Refs. 2Houtkooper R.H. Vaz F.M. Cell. Mol. Life Sci. 2008; 65: 2493-2506Crossref PubMed Scopus (304) Google Scholar, 7Chicco A.J. Sparagna G.C. Am. J. Physiol. Cell Physiol. 2007; 292: C33-C44Crossref PubMed Scopus (498) Google Scholar, 11Hauff K.D. Hatch G.M. Prog. Lipid Res. 2006; 45: 91-101Crossref PubMed Scopus (123) Google Scholar, 12Schlame M. Ren M. FEBS Lett. 2006; 580: 5450-5455Crossref PubMed Scopus (250) Google Scholar). In addition, the role that CL plays in apoptosis is now well documented (reviewed in Ref. 13Ott M. Zhivotovsky B. Orrenius S. Cell Death Differ. 2007; 14: 1243-1247Crossref PubMed Scopus (164) Google Scholar). Thus, maintenance of the appropriate content and fatty acyl composition of CL in mitochondria is essential for proper cellular function. cardiolipin monolysocardiolipin monolysocardiolipin acyltransferase tafazzin matrix activated laser desorption ionization-time of flight Barth syndrome acyllysocardiolipin acyltransferase-1 monolysocardiolipin acyltransferase-1 endoplasmic reticulum RNA interference. The molecular composition of CL appears to be important for the biological function of CL. In general, there is a selection of a particular kind of fatty acid as well as restriction of the number of fatty acid species (14Schlame M. Ren M. Xu Y. Greenberg M.L. Haller I. Chem. Phys. Lipids. 2005; 138: 38-49Crossref PubMed Scopus (235) Google Scholar). The major tetra-acyl molecular species found in rat liver (∼57% of total) and bovine heart (∼48% of total) are 18:2 in each of the four fatty acyl positions of the cardiolipin molecule. Remodeling of CL is essential to obtain this enrichment of CL with linoleate because CL synthase has no molecular species substrate specificity for cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol (15Hostetler K.Y. Galesloot J.M. Boer P. Van Den Bosch H. Biochim. Biophys. Acta. 1975; 380: 382-389Crossref PubMed Scopus (72) Google Scholar). In addition, the species pattern of CL precursors is similar enough to imply that the enzymes of the CL synthetic pathway are not molecular species-selective (16Rüstow B. Schlame M. Rabe H. Reichmann G. Kunze D. Biochim. Biophys. Acta. 1989; 1002: 261-263Crossref PubMed Scopus (35) Google Scholar). Alterations in the molecular composition of CL are associated with various disease states, including diabetes and Barth syndrome (17Han X. Yang J. Yang K. Zhao Z. Abendschein D.R. Gross R.W. Biochemistry. 2007; 46: 6417-6428Crossref PubMed Scopus (224) Google Scholar, 18Valianpour F. Wanders R.J. Barth P.G. Overmars H. van Gennip A.H. Clin. Chem. 2002; 48: 1390-1397Crossref PubMed Scopus (104) Google Scholar). Remodeling of CL occurs via at least three enzymes. Mitochondrial CL was shown to be remodeled by a deacylation-reacylation cycle in which newly synthesized CL was rapidly deacylated to MLCL and then reacylated back to CL with linoleoyl-CoA (19Schlame M. Rüstow B. Biochem. J. 1990; 272: 589-595Crossref PubMed Scopus (104) Google Scholar). A mitochondrial MLCL acyltransferase (MLCL AT) activity was characterized and purified from pig liver mitochondria (20Ma B.J. Taylor W.A. Dolinsky V.W. Hatch G.M. J. Lipid Res. 1999; 40: 1837-1845Abstract Full Text Full Text PDF PubMed Google Scholar, 21Taylor W.A. Hatch G.M. J. Biol. Chem. 2003; 278: 12716-12721Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). An acyl-CoA-dependent reacylation of MLCL to CL was shown to occur in rat liver microsomes (22Eichberg J. J. Biol. Chem. 1974; 249: 3423-3429Abstract Full Text PDF PubMed Google Scholar). This enzyme was identified as acyllysocardiolipin acyltransferase-1 (ALCAT1) (23Cao J. Liu Y. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2004; 279: 31727-31734Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Recently it was shown that ALCAT1 expression in endothelial and hematopoietic lineages resulted in elevated hematopoietic and endothelial genes and increased blast colonies and their progenies (24Wang C. Faloon P.W. Tan Z. Lv Y. Zhang P. Ge Y. Deng H. Xiong J.W. Blood. 2007; 110: 3601-3609Crossref PubMed Scopus (34) Google Scholar, 25Xiong J.W. Yu Q. Zhang J. Mably J.D. Circ. Res. 2008; 102: 1057-1064Crossref PubMed Scopus (50) Google Scholar). The opposite effect was observed with ALCAT1 small interfering RNA indicating that ALCAT1 may play a role in the early specification of hematopoietic and endothelial cells (24Wang C. Faloon P.W. Tan Z. Lv Y. Zhang P. Ge Y. Deng H. Xiong J.W. Blood. 2007; 110: 3601-3609Crossref PubMed Scopus (34) Google Scholar, 25Xiong J.W. Yu Q. Zhang J. Mably J.D. Circ. Res. 2008; 102: 1057-1064Crossref PubMed Scopus (50) Google Scholar). In addition to these mitochondrial and microsomal acyltransferase activities, mitochondrial CL may be remodeled by a mitochondrial CL transacylase reaction first described in rat liver (26Xu Y. Kelley R.I. Blanck T.J. Schlame M. J. Biol. Chem. 2003; 278: 51380-51385Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The Barth syndrome gene product tafazzin (TAZ) is a CL transacylase (27Xu Y. Malhotra A. Ren M. Schlame M. J. Biol. Chem. 2006; 281: 39217-39224Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Although TAZ specifically remodels mitochondrial CL with linoleic acid, TAZ alone cannot determine the fatty acid profile of mitochondrial CL (3Schlame M. J. Lipid. Res. 2008; 49: 1607-1620Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). In this study, we identify a human protein, MLCL AT-1, with a linoleoyl coenzyme A-specific mitochondrial MLCL AT activity. [1-14C]Linoleic acid, [1-14C]linoleoyl coenzyme A, [1-14C]oleoyl coenzyme A, [1-14C]oleic acid, [1-14C]palmitoyl coenzyme A, and [1-14C]palmitic acid were obtained from either DuPont or Amersham Biosciences or American Radiolabeled Chemicals Inc., St. Louis, MO. Dulbecco's modified Eagle's medium and fetal bovine serum were products of Invitrogen. Lipid standards were obtained from Serdary Research Laboratories, Englewood Cliffs, NJ. MLCL was obtained from Avanti Polar Lipids, Alabaster, NY. Thin layer chromatographic plates (silica gel G, 0.25-mm thickness) were obtained from Fisher. Ecolite scintillant was obtained from ICN Biochemicals, Montreal, Quebec, Canada. HeLa cells were obtained from American Type Culture Collection. Epstein-Barr virus-transformed BTHS lymphoblasts (patient 596) were a generous gift from Dr. Richard Kelley, The John Hopkins University. Epstein-Barr virus-transformed age-matched control lymphoblasts were obtained from Coriell Institute for Medical Research, Camden, NJ. Western blotting analysis system was used for protein expression studies and was obtained from Amersham Biosciences. Kodak X-Omat film was obtained from Eastman Kodak Co. Qiagen OneStep RT-PCR kit was used for PCR studies. All other chemicals were certified ACS grade or better and obtained from Sigma or Fisher. Three kg of pig liver from the local meat packers were homogenized in buffer A (10 mm Tris, 0.25 m sucrose, 2 mm EDTA, and 10 mm 2-mercaptoethanol) using a Polytron at medium speed for 5 min. The extract was then centrifuged at 2000 × g for 20 min to remove cell membranes and nuclei. The supernatant was again centrifuged at 8,500 rpm for 55 min to isolate the crude mitochondria. One liter of ice-cold buffer A was added and kept at 4 °C for 3 days with intermittent stirring at low speed with a Polytron homogenizer. Thereafter, the extract was centrifuged at 12,000 × g to produce a clarified mitochondrial extract (675 ml) that was concentrated to 170 ml by Hollow Fiber (Amicon) concentration and then freeze-dried. The freeze-dried extract was treated with 500 ml of butanol with stirring as described (28Morton R.K. Methods Enzymol. 1955; 1: 26-51Google Scholar). The mitochondrial extract was then subjected to anion exchange chromatography with a Q-Sepharose column that was washed thoroughly with buffer A containing 0.2 m NaCl. MLCL AT enzyme activity was eluted from the column with 0.3 mm acetyl coenzyme A in buffer A containing 0.2 m NaCl. This acetyl coenzyme A fraction was dialyzed, freeze-dried, and then applied to a preparatory gel electrophoresis column containing 5% acrylamide. The proteins were separated on the gel with 40 mA constant current with buffer A as the elution buffer. The fractions containing MLCL AT activity were pooled and then applied to a cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose gel and washed with buffer A containing 0.2 m NaCl. The fraction containing MLCL AT activity was eluted with 5 mm CTP and 0.2 m NaCl in buffer A. The eluted fraction was dialyzed in buffer A and then added to an MLCL-adriamycin affinity column (see below). The MLCL-adriamycin affinity resin was prepared by mixing 30 ml of REACTI-gel (Pierce) with 1.2 mg of adriamycin in 45 ml of 0.1 m borate buffer at 4 °C. Before addition of the partially pure MLCL AT, the gel was mixed with 1 mg of MLCL and washed with 0.1 m borate, pH 8.0. The eluted fraction from above was applied to the MLCL-adriamycin-agarose and washed thoroughly with 0.1 m borate, pH 8.0, until there was no more protein emerging from the column. Finally the MLCL AT activity was eluted from the affinity column with borate buffer containing 1 mm linoleoyl coenzyme A or 1 mm MLCL. A polyvinyl difluoride membrane blot was submitted for MALDI-TOF-mass spectrometry analysis (University of Minnesota). The peptide sequences obtained were analyzed by searching the Mascot protein data base for identification of potential homologies with other known proteins. An NCBI BLAST search for known proteins that align with the peptide sequences was performed using the Protein Data Bank. The full-length primers for the human 59-kDa unknown protein (GenBankTM accession number AC011742.3) MLCL AT-1 containing a His6 tag in the reverse primer without stop codon was prepared from Invitrogen (custom primer design) (Table 1). The His tag was required for binding of the protein to the nickel-nitrilotriacetic acid affinity resin (see below). The primers were amplified using 1 μg of HeLa cell RNA. The cDNAs containing the full-length sequences were inserted into pcDNA 3.1 using the TOPO cloning reaction with pEXP5-CT/TOPO vector (Invitrogen). Escherichia coli One Shot® bacteria (Invitrogen) were transformed with the construct chemically with S.O.C. medium (Invitrogen) and inoculated onto ampicillin containing agar for growth overnight. In the morning, the colonies were inoculated into 5 ml of ampicillin containing LB medium and cultured at 37 °C in an orbital shaker at 220 rpm overnight. The plasmid was purified from the E. coli using the MidiPrep kit (Invitrogen). The sequences of the plasmids were verified by PCR using the specific primers and also by a DNA sequencer (Manitoba Institute of Cell Biology). The recombinant protein was expressed using the cell-free E. coli expression system (Invitrogen). The recombinant protein was purified with a nickel-nitrilotriacetic acid affinity resin (Fisher) when eluted with 200 mm imidazole. When high purity proteins were required, the resin was first of all pre-eluted with high salt (1 m NaCl) and low imidazole concentration (20 mm).TABLE 1Primers used for in vitro protein expression and transfection The cDNA containing the full-length sequence of the human unknown protein, MLCL AT-1, was inserted into pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen) and grown in E. coli, and the plasmid was purified and sequence-verified as described above. The plasmid was used for transfection of HeLa cells or BTHS lymphoblasts as described below. RNAi to human MLCL AT-1 was prepared from Invitrogen using the BLOCK-iT RNAi Designer program (Table 1). The RNAi was used for transfection of HeLa cells or lymphoblasts as described below. HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. To evaluate the effect of the human unknown protein on CL acylation with different species of fatty acid, 13 μg of MLCL AT-1 protein plasmid in 23 μl of Lipofectamine (Invitrogen) were added to HeLa cells at 50% confluence. After 24 h, 1 μCi of fatty acid ([1-14C]linoleic acid, [1-14C]oleic acid, or [1-14C]palmitic acid (bound to bovine serum albumin in a 1:1 molar ratio) was added and incubation continued for another 24 h. Cells were then harvested, and radioactivity was incorporated into CL determined as described previously (28Morton R.K. Methods Enzymol. 1955; 1: 26-51Google Scholar). In other experiments, 100 nm of RNAi in 23 μl of Lipofectamine (Invitrogen) were added to HeLa cells at 50% confluence. After 24 h, 1 μCi of [1-14C]linoleic acid was added and incubation continued for another 24 h, and radioactivity was incorporated into CL determined as described above. In some experiments, cells were washed twice with ice-cold saline and harvested with 2 ml of lysis buffer (10 mm Tris-HCl, pH 7.4, 0.25 m sucrose) and then homogenized with 30 strokes of a Dounce A homogenizer. The homogenate was centrifuged at 1,000 × g for 5 min and the supernatant centrifuged at 10,000 × g for 15 min. The pellet was resuspended in 0.5 ml of homogenization buffer and used for assay of mitochondrial enzyme activities as described below. BTHS lymphoblasts and age-matched control lymphoblasts were grown in suspension in RPMI 1640 medium containing 10% fetal bovine serum until reaching a concentration of 106 cells/ml. Lymphoblasts were pelleted and placed in Opti-MEM (Invitrogen) (5 × 106 cells/ml) and incubated with 40 μg of MLCL AT-1 plasmid, and electroporation was performed at 950 microfarads, 250 V, for 23 ms in 800 μl of Opti-MEM using a BTX Electroporation System Electrocell Manipulator 600. Cells were then incubated with 1 μCi [1-14C]linoleic acid for 4 h, and radioactivity was incorporated into CL determined as described above. In other experiments mitochondria from the above cells were prepared as above, and MLCL AT or succinate dehydrogenase activities were determined as described below. In other experiments, 100 nm small interfering RNA was transfected into lymphocytes (5 × 106 cells/ml) by electroporation as described above. Following electroporation the lymphocytes were incubated in 10 ml of Opti-MEM for 19 h. Subsequently the cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% each of antimycotic and antibiotic. Incubation at 37 °C and 5% CO2 was continued for an additional 24 h following which the cells were harvested and the mitochondria isolated as described previously. In other experiments, BTHS lymphoblasts were incubated as above, and CL was isolated and a phosphorous mass of CL determined as described previously (29Hatch G.M. McClarty G. J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). MLCL AT activity was determined as described previously (20Ma B.J. Taylor W.A. Dolinsky V.W. Hatch G.M. J. Lipid Res. 1999; 40: 1837-1845Abstract Full Text Full Text PDF PubMed Google Scholar, 21Taylor W.A. Hatch G.M. J. Biol. Chem. 2003; 278: 12716-12721Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Essentially human recombinant MLCL AT-1 protein (20 ng) or mitochondrial protein (20 μg) was incubated in 50 mm Tris-HCl buffer, pH 8.0, and incubated with 0.3 mm MLCL and [1-14C]linoleoyl coenzyme A (120,000 dpm/nmol) at 37 °C for 1 h or longer for smaller protein quantities. For some reactions [1-14C]linoleoyl coenzyme A was replaced with [1-14C]oleoyl coenzyme A or [1-14C]palmitoyl coenzyme A. The reaction was stopped by the addition of chloroform/methanol (2:1). The organic fraction was isolated by centrifuging the mixture after the addition of 0.9% KCl. After an additional washing of the organic fraction with theoretical upper phase, the chloroform layer was dried with nitrogen, resuspended in 25 μl of chloroform/methanol (2:1), and applied to a Whatman silica gel-coated glass thin layer plate with CL standard. [14C]CL was isolated by two-dimensional chromatography using the following solvent mixtures: first dimension (chloroform/methanol/water, 65:25:4, by volume) and second dimension (chloroform/acetone/methanol/acetic acid/water, 50:20:10:10:5, by volume). CL was visualized with iodine vapor and silica gel corresponding to the CL spot removed and placed into scintillation vials containing 5 ml of Ecolite scintillant, and radioactivity was determined in an LS 6500 liquid scintillation counter (Beckman). For kinetic analysis in some experiments MLCL AT activity of the recombinant human MLCL AT-1 protein was determined in the presence of a fixed amount of [1-14C]acyl coenzyme A with varying concentrations of MLCL and/or a fixed amount of MLCL with varying concentrations of [1-14C] acyl coenzyme A, and the reciprocal velocity versus substrate concentration was plotted. Mitochondrial succinate dehydrogenase activity was determined as described (30Singer T.P. Methods Biochem. Anal. 1974; 22: 123-175Crossref PubMed Google Scholar). Proteins from the MLCL-adriamycin affinity column eluted with either linoleoyl coenzyme A or MLCL as described above were separated on the Bio-Rad mini gel electrophoresis system using 10% acrylamide containing 0.1% SDS. The disruption buffer (1×, Sigma) included SDS, 2-mercaptoethanol, and bromphenol blue dye. The electrophoresis was performed using synthetic pre-stained molecular markers from Bio-Rad. After the electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes, using Tris-glycine buffer, pH 8.3, with 20% methanol, at 15 V for 1.5 h. The proteins were probed overnight with polyclonal pig liver anti-MLCL AT antibody (21Taylor W.A. Hatch G.M. J. Biol. Chem. 2003; 278: 12716-12721Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The second antibody was anti-rabbit IgG. The protein was visualized on X-Omat film by chemiluminescence (Amersham Biosciences). In other experiments, 0.5 or 1 μg of recombinant protein from the in vitro protein translation or 12 μg of HeLa cell mitochondrial protein or 1.2 μg of lymphoblast mitochondrial protein was separated on a 10% SDS-polyacrylamide gel, blotted onto a polyvinylidene difluoride membrane, and probed with anti-MLCL AT-1 antibody as above. The MLCL-AT-1 was visualized by chemiluminescence, and the protein gel was stained with Coomassie Blue. Protein was determined as described (31Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Student's t test was used for determination of statistical significance. The level of significance was defined as p < 0.05. To identify the human mitochondrial MLCL AT, a pig liver mitochondrial extract was prepared as described under “Experimental Procedures.” Following elution from the cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose gel the MLCL AT activity containing sample was dialyzed and mixed with MLCL-adriamycin-agarose and then applied to a column and extensively washed with 0.2 m NaCl in 0.1 m borate buffer, pH 9.0. MLCL AT enzyme activity was eluted with 1.0 mm MLCL (Fig. 1A). The specific activity of fraction 10 was 2,306 pmol/min/mg protein. Western blot analysis of fraction 10 using a polyclonal antibody to the pig liver MLCL AT demonstrated the presence of a protein at 74 kDa (Fig. 1B). In addition, the identical amount of MLCL AT activity could be eluted in fraction 10 with 1.0 mm linoleoyl coenzyme A. Western blot analysis of fraction 10 eluted by linoleoyl-CoA demonstrated the presence of a protein at 74 kDa (Fig. 1B). MALDI-TOF-mass spectrometry analysis of the 74-kDa protein revealed peptide matches to enoyl-coenzyme A hydratase, NAD-binding protein containing the Rossmann-fold for NAD(P) binding, and coenzyme A dehydrogenase (Table 2). These motifs are consistent with the 74-kDa pig liver mitochondrial trifunctional protein α (GenBankTM accession number NP_999127). Further peptide analysis indicated the presence of motifs for lipid, coenzyme A, and acyl coenzyme A binding and acyltransferase activity (Table 3). Alignment of human mitochondrial trifunctional protein α (GenBankTM accession number NP_000173) revealed a match with a 59-kDa human unknown protein (GenBankTM protein accession number AAX93141; nucleotide accession number AC011742.3) (Fig. 2). AAX93141 is identical to the C-terminal 59-kDa end of mitochondrial trifunctional protein. Proteins (GenBankTM accession numbers 3BLN_A; 1B3O_A; 2OME_A; and 1IVH_A) identified from the blast search were then aligned using the Cobalt multialignment program in comparison with the NP_000173 and AAX93141 (Fig. 2). Identical amino acids are highlighted, and the location of the peptides and amino acid identities are represented in boldface with the region of agreement underlined (Fig. 2).TABLE 2Trifunctional protein motifs obtained from MALDI-TOF-mass spectrometry and BLAST search protein matchesTABLE 2Trifunctional protein motifs obtained from MALDI-TOF-mass spectrometry and BLAST search protein matchesTABLE 3Peptide sequences obtained from MALDI-TOF-mass spectrometry and BLAST search protein matchesTABLE 3Peptide sequences obtained from MALDI-TOF-mass spectrometry and BLAST search protein matchesFIGURE 2Sequence alignment of AAX93141 with proteins identified through peptide matches. Proteins (3BLN_A, 1B3O_A, 2OME_A, and 1IVH_A) obtained from the BLAST search of the peptides and human trifunctional protein (NP_000173) were analyzed by the Cobalt multialignment program. The amino acid residues of the known proteins are highlighted (gray) relative to their alignment with the human MLCL AT-1 (AAX93141) and NP_000173. Matching amino acid sequences from peptides I to V from Table 3 are indicated in boldface. Protein accession number is indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the MLCL AT activity of the mitochondrial 74-kDa protein had been characterized previously (21Taylor W.A. Hatch G.M. J. Biol. Chem. 2003; 278: 12716-12721Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), purified human recombinant 59-kDa mitochondrial trifunctional protein (protein accession number AAX93141) was prepared as outlined under “Experimental Procedures” Western blot analysis of the protein exhibited cross-reaction with the 74-kDa pig liver MLCL AT polyclonal antibody at 59 kDa (Fig. 3A). The recombinant protein was then further purified following elution from a nickel resin. SDS-PAGE analysis of the purified recombinant protein revealed the 59-kDa protein as a single band (Fig. 3B). Thus, only the 59-kDa protein was present following elution from the nickel resin. Two μg of the purified protein was examined for the ability to acylate MLCL to CL in the presence of [1-14C]linoleoyl coenzyme A. The eluate from the nickel resin prepared from the recombinant protein could acylate MLCL to CL in the presence of [1-14C]linoleoyl coenzyme A (Fig. 3C). No MLCL AT activity was observed in preparations that did not contain expression vector in the in vitro translation system. Thus, the 59-kDa human protein exhibits MLCL AT activity and was termed MLCL" @default.
• W2006966998 created "2016-06-24" @default.
• W2006966998 creator A5026563745 @default.
• W2006966998 creator A5057844469 @default.
• W2006966998 date "2009-10-01" @default.
• W2006966998 modified "2023-09-30" @default.
• W2006966998 title "Identification of the Human Mitochondrial Linoleoyl-coenzyme A Monolysocardiolipin Acyltransferase (MLCL AT-1)" @default.
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