Cloning and characterization of a cDNA encoding human cardiolipin synthase (hCLS1)

Cardiolipin (CL) is a phospholipid localized to the mitochondria, and its biosynthesis is essential for mitochondrial structure and function. We report here the identification and characterization of a cDNA encoding the first mammalian cardiolipin synthase (CLS1) in humans and mice. This cDNA exhibits sequence homology with members of a CLS gene family that share similar domain structure and chemical properties. Expression of the human CLS (hCLS1) cDNA in reticulocyte lysates or insect cells led to a marked increase in CLS activity. The enzyme is specific for CL synthesis, because no significant increase in phosphatidylglycerol phosphate synthase activity was observed. In addition, CL pool size was increased in hCLS1-overexpressing cells compared with controls. Furthermore, the hCLS1 gene was highly expressed in tissues such as heart, skeletal muscle, and liver, which have been shown to have high CLS activities. These results demonstrate that hCLS1 encodes an enzyme that synthesizes CL. Cardiolipin (CL) is a phospholipid localized to the mitochondria, and its biosynthesis is essential for mitochondrial structure and function. We report here the identification and characterization of a cDNA encoding the first mammalian cardiolipin synthase (CLS1) in humans and mice. This cDNA exhibits sequence homology with members of a CLS gene family that share similar domain structure and chemical properties. Expression of the human CLS (hCLS1) cDNA in reticulocyte lysates or insect cells led to a marked increase in CLS activity. The enzyme is specific for CL synthesis, because no significant increase in phosphatidylglycerol phosphate synthase activity was observed. In addition, CL pool size was increased in hCLS1-overexpressing cells compared with controls. Furthermore, the hCLS1 gene was highly expressed in tissues such as heart, skeletal muscle, and liver, which have been shown to have high CLS activities. These results demonstrate that hCLS1 encodes an enzyme that synthesizes CL. Cardiolipin (CL) is a phospholipid localized to the mitochondria (1Stoffel W. Schiefer H.G. Biosynthesis and composition of phosphatides in outer and inner mitochondrial membranes.Hoppe Seylers Z. Physiol. Chem. 1968; 349: 1017-1026Google Scholar), where it plays a key role in mitochondrial function (2Vik S.B. Georgevich G. Capaldi R.A. Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase.Proc. Natl. Acad. Sci. USA. 1981; 78: 1456-1460Google Scholar, 3Gohil V.M. Hayes P. Matsuyama S. Schagger H. Schlame M. Greenberg M.L. Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent.J. Biol. Chem. 2004; 279: 42612-42618Google Scholar). In eukaryotic cells, CL is predominantly present in mitochondrial inner membranes (4Schlame M. Rua D. Greenberg M.L. The biosynthesis and functional role of cardiolipin.Prog. Lipid Res. 2000; 39: 257-288Google Scholar, 5van Klompenburg W. Nilsson I. von Heijne G. de Kruijff B. Anionic phospholipids are determinants of membrane protein topology.EMBO J. 1997; 16: 4261-4266Google Scholar), where it provides osmotic stability to membranes and interacts with several key mitochondrial enzymes for their optimal oxidative activities (6Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.J. Biol. Chem. 2000; 275: 22387-22394Google Scholar, 7Zhao M. Schlame M. Rua D. Greenberg M.L. Cardiolipin synthase is associated with a large complex in yeast mitochondria.J. Biol. Chem. 1998; 273: 2402-2408Google Scholar). In mammals, CL is thought to be required for many functions. For example, alterations in the content and the acyl composition of CL result in a mitochondrial structural abnormality (8Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome.Biochem. Biophys. Res. Commun. 2000; 279: 378-382Google Scholar, 9Ohtsuka T. Nishijima M. Suzuki K. Akamatsu Y. Mitochondrial dysfunction of a cultured Chinese hamster ovary cell mutant deficient in cardiolipin.J. Biol. Chem. 1993; 268: 22914-22919Google Scholar) and reduced oxygen consumption (10Ohtsuka T. Nishijima M. Akamatsu Y. A somatic cell mutant defective in phosphatidylglycerophosphate synthase, with impaired phosphatidylglycerol and cardiolipin biosynthesis.J. Biol. Chem. 1993; 268: 22908-22913Google Scholar). Moreover, a reduction in CL is a key abnormality both in Barth syndrome (11Bolhuis P.A. Hensels G.W. Hulsebos T.J. Baas F. Barth P.G. Mapping of the locus for X-linked cardioskeletal myopathy with neutropenia and abnormal mitochondria (Barth syndrome) to Xq28.Am. J. Hum. Genet. 1991; 48: 481-485Google Scholar), an X-linked genetic disease, and in animal models (12Hagen T.M. Ingersoll R.T. Wehr C.M. Lykkesfeldt J. Vinarsky V. Bartholomew J.C. Song M.H. Ames B.N. Acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity.Proc. Natl. Acad. Sci. USA. 1998; 95: 9562-9566Google Scholar, 13Petrosillo G. Ruggiero F.M. Paradies G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria.FASEB J. 2003; 17: 2202-2208Google Scholar). Although the exact role that CL plays in the apoptotic process remains controversial (14Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane.Cell. 2002; 111: 331-342Google Scholar, 15Iverson S.L. Enoksson M. Gogvadze V. Ott M. Orrenius S. Cardiolipin is not required for Bax-mediated cytochrome c release from yeast mitochondria.J. Biol. Chem. 2004; 279: 1100-1107Google Scholar), data suggest that alterations in CL metabolism are involved in regulating mammalian aging (12Hagen T.M. Ingersoll R.T. Wehr C.M. Lykkesfeldt J. Vinarsky V. Bartholomew J.C. Song M.H. Ames B.N. Acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity.Proc. Natl. Acad. Sci. USA. 1998; 95: 9562-9566Google Scholar) and cell death (16Ostrander D.B. Sparagna G.C. Amoscato A.A. McMillin J.B. Dowhan W. Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis.J. Biol. Chem. 2001; 276: 38061-38067Google Scholar). Thus, maintenance of CL is required for mammalian cell function. In mammalian tissue, CL and its synthetic enzyme cardiolipin synthase (CLS) are located exclusively in mitochondria. De novo CL biosynthesis occurs on the matrix side of the inner mitochondrial membrane (17Schlame M. Haldar D. Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria.J. Biol. Chem. 1993; 268: 74-79Google Scholar) via the cytidine-5′diphosphate-1,2-diacylglycerol (CDP-DG) pathway [reviewed in (4Schlame M. Rua D. Greenberg M.L. The biosynthesis and functional role of cardiolipin.Prog. Lipid Res. 2000; 39: 257-288Google Scholar)]. Phosphatidic acid is converted to CDP-DG by CDP-DG synthase. The CDP-DG formed then condenses with glycerol-3-phosphate to form phosphatidylglycerol (PG) catalyzed by phosphatidylglycerol phosphate (PGP) synthase and PGP phosphatase. In the final step, PG is converted to CL by condensation with CDP-DG catalyzed by the terminal enzyme CLS. Although a mammalian CLS has been purified to homogeneity (18Schlame M. Hostetler K.Y. Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement.J. Biol. Chem. 1991; 266: 22398-22403Google Scholar), its gene has not been identified. Through homology searches of the expressed sequence tag (EST) databases with the yeast CLS, we identified a UniGene on human chromosome 20, (C20orf155). In this study, we demonstrate that C20orf155 encodes a human CLS, now termed hCLS1. The expression of hCLS1 cDNA in vitro and in vivo resulted in high levels of CLS activity, supporting its role as a CLS. [α-32P]dCTP was purchased from Perkin-Elmer Life Sciences (Boston, MA). TLC (silica gel 60) plates were from Fisher Scientific. Lipid standards were from Serdary Research Laboratories. All other biochemicals were from Sigma or Fisher Scientific. Insect Sf9 cells were from Invitrogen. Cells were grown in 10 cm dishes in Grace’s Insect Medium supplemented with 10% fetal calf serum at 27°C. For transfection, recombinant viral DNA in Cellfectin Reagent was added to Sf9 cells according to the Baculovirus Expression Systems protocol (Invitrogen). After transfection, P1 viral stock was collected. Northern blots containing poly(A) RNA from selected human tissues were purchased from Clontech (Palo Alto, CA). Hybridization was performed using [α-32P]dCTP-labeled hCLS1 probes as described (19Lu B. Kelher M.R. Lee D.P. Lewin T.M. Coleman R.A. Choy P.C. Hatch G.M. Complex expression pattern of the Barth syndrome gene product tafazzin in human cell lines and murine tissues.Biochem. Cell Biol. 2004; 82: 569-576Google Scholar). The coding region of hCLS1 was amplified with a pair of specific primers (Fig. 1A). PCR was performed using Platinum Pfx DNA polymerase (Invitrogen). Sequencing analysis was performed at the Institute of Cell Biology in Winnipeg, Canada.Fig. 1The human cardiolipin synthase protein (hCLS1) and its tissue expression pattern. A: Features of the predicted peptide sequence of hCLS1. A mitochondrial targeting sequence at the N terminus is shaded. The region conserved among proteins containing a CDP-alcohol phosphatidyltransferase domain (KOG1617; pfam01066) or a PGP synthase domain (COG0558) is underlined. The poly(A) signal is indicated with asterisks. The arrow indicates the cleavage and a poly(A) tail. The boxed nucleotide sequences are the primers used to amplify the coding cDNA. B: Tissue expression pattern of hCLS1. Human multiple tissue blots containing 2 μg of poly(A)+ RNA for each tissue were hybridized with radiolabeled probes prepared from the full-length cDNA of hCLS1. The hybridized blots were stripped of residual radioactivity and reprobed with β-actin cDNA as an internal control.View Large Image Figure ViewerDownload (PPT) Yeast CLS protein sequences were used to search against the translated est_human database. More than 100 BLAST hits revealed one unique gene, C20orf155. One EST clone (clone identifier 2369931; Invitrogen) containing hCLS1 cDNA was purchased and sequenced. The full-length coding sequences were amplified using Platinum Pfx DNA polymerase (Invitrogen). The amplified DNA fragment was subcloned into an expression vector, pcDNA3.1/V5-His (Invitrogen). Similarly, mouse CLS (mCLS1) cDNA was obtained and engineered into pcDNA3.1/V5-His vector. The in vitro transcription and translation reactions were performed using a coupled transcription and translation system (TnT Coupled Reticulocyte Lysate System; Promega), as described previously (20Lu B. Jiang Y.J. Zhou Y. Xu F.Y. Hatch G.M. Choy P.C. Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.Biochem. J. 2005; 385: 469-477Google Scholar). The hCLS1/mCLS1 sequences were first subcloned into pENTR and subsequently into baculovirus vector according to the manufacturer's manual (Invitrogen). Recombinant viruses were generated by transfecting Sf9 cells and selecting with ganciclovir. High viral titers were obtained from three rounds of amplification. P3 recombinant viruses were used to express recombinant hCLS1/mCLS1 in Sf9 cells. Samples containing ∼10–20 μl of reticulocyte lysates or ∼80–100 μg of cell extracts from virus-infected Sf9 cells were subjected to SDS-PAGE as described (20Lu B. Jiang Y.J. Zhou Y. Xu F.Y. Hatch G.M. Choy P.C. Cloning and characterization of murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in murine heart.Biochem. J. 2005; 385: 469-477Google Scholar). The protein fractions in the gel were transferred onto polyvinylidene difluoride membranes, which were incubated with conjugated anti-V5 antibodies (1:5,000 dilution; Invitrogen). The protein bands were detected using the SuperSignal Extended Duration Substrate Kit (Pierce, Rockford, IL). PGP synthase and CLS enzyme assays were measured using methods described previously (21Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Google Scholar). The pool sizes of major phospholipids were determined in control and hCLS1-overexpressing Sf9 cells as described (21Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Google Scholar). Results are presented as means ± SEM. Statistical significance between two groups was determined by Student’s t-test. P < 0.05 was considered significant. Through homology searches of the EST databases using yeast CLS, we identified a cDNA encoding putative hCLS1. The translation of hCLS1 predicts a 301 amino acid protein that exhibits 34% sequence identity to yeast CLS sequences (162 amino acids near the C terminus) (Fig. 1A). Analysis of hCLS1 protein sequences revealed two critical features. At its N terminus, hCLS1 exhibited a preponderance of basic and hydroxyl-carrying residues, suggesting mitochondrial target signaling (22Emanuelsson O. Nielsen H. Brunak S. von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.J. Mol. Biol. 2000; 300: 1005-1016Google Scholar) (Fig. 1A). This mitochondrial target signaling of hCLS1 was also predicted by a subcellular localization prediction program (TargetP1.1 server at http://www.cbs.dtu.dk/cgi-bin/). At its C terminus, a significant sequence alignment with both CDP-alcohol phosphatidyltransferase (pfam01066; CD-Length = 145 residues, 83.4% aligned) and PGP synthase (COG0558; CD-Length = 192 residues, 80.7% aligned) was revealed, suggesting an enzyme containing CDP-alcohol phosphatidyltransferase activity (Fig. 1A). The hCLS1 EST sequences were mapped to a region of human chromosome 20, C20orf155 (GenBank accession number 27501067). Because the EST sequences are mapped to the chromosome DNA, hCLS1 is transcribed from human genome, but not mitochondrial genome. As shown in Fig. 1B, the hCLS1 probe hybridized primarily with two dominant mRNA transcripts at ∼1.6 and ∼2.2 kb as well as with a minor transcript at ∼4.1 kb. hCLS1 was highly expressed in heart, skeletal muscle, and liver; low to moderate levels were seen in pancreas, kidney, and placenta; and very low levels were found in lung and brain. This tissue expression pattern correlates well with CLS activity in these tissues (4Schlame M. Rua D. Greenberg M.L. The biosynthesis and functional role of cardiolipin.Prog. Lipid Res. 2000; 39: 257-288Google Scholar). Through homology searches of nonredundant databases at the National Center for Biotechnology Information, the mouse (AAH48702) and rat (NP_0014280) CLS sequences were also identified. rCLS1 and mCLS1 shared 85% and 86% amino acid sequence identity with hCLS1, fulfilling the criteria for counterparts in different species. To define the sequence conservation among CLS family members, eukaryotic CLS proteins were analyzed with the ClustalW alignment program (Fig. 2A). Although the overall matches between members varied (21–97%), the region near the protein C terminus is largely conserved. Within this region, four highly conserved stretches/motifs were apparent: a CDP-OH-P motif, D(X)2DG(X)2AR(X)8-9G(X)3D(X)3D(X)2L, which is necessary for CLS enzyme activity (23Katayama K. Sakurai I. Wada H. Identification of an Arabidopsis thaliana gene for cardiolipin synthase located in mitochondria.FEBS Lett. 2004; 577: 193-198Google Scholar), and three other novel motifs that may be important for CLS structure and function (23Katayama K. Sakurai I. Wada H. Identification of an Arabidopsis thaliana gene for cardiolipin synthase located in mitochondria.FEBS Lett. 2004; 577: 193-198Google Scholar). Because all eukaryotic CLS proteins identified to date have multiple transmembrane domains at the C terminus, we examined whether hCLS1 also has this feature. Analysis by transmembrane domain programs (http://www.cbs.dtu.dk/services/TMHMM/) found two hydrophobic domains (amino acid positions 120–138 and 178–189) and three transmembrane helices (amino acid positions 190–212, 240–262, and 267–289) (Fig. 2B). This topology is consistent with previous findings that CLS has multiple membrane domains overlapping its catalytic region (23Katayama K. Sakurai I. Wada H. Identification of an Arabidopsis thaliana gene for cardiolipin synthase located in mitochondria.FEBS Lett. 2004; 577: 193-198Google Scholar).Fig. 2Sequence comparison of members of the CLS gene family. A: Alignment of predicted mammalian CLS protein sequences (human, rat, mouse) with those of plant and yeast CLS. The protein sequences were from hCLS1 (NP_061968), mouse CLS1 (mCLS1; AAH48702), rat CLS1 (rCLS1; NP_0014280), plant Arabidopsis thaliana CLS (AtCLS; NP_567273), and yeast Saccharomyces cerevisiae CLS (ScCLS; NP_010139.1). Dashes represent gaps to maximize alignment of the sequences. Amino acid residues identical for all eukaryotic CLS proteins are indicated with asterisks. Conservation of conserved substitutions is indicated with two dots, and semiconserved substitutions are indicated with one dot. The amino acid residues conserved with the CDP-OH-P motif are shaded in black. Four novel conserved motifs are underlined. B: Hydrophobicity plot of hCLS1 as assessed by Kyte-Doolittle analysis. The thick solid lines indicate three predicted transmembrane helices (TM1–TM3), and the thick dashed lines indicate two membrane binding regions (MB1 and MB2).View Large Image Figure ViewerDownload (PPT) To examine whether our cloned mammalian CLS1 has the ability to encode a protein, mCLS1 and hCLS1 were expressed in two experimental systems. First, expression of both V5-His-tagged mCLS1 and hCLS1 was performed in rabbit reticulocyte lysates using an in vitro coupled transcription and translation system. Expression of tagged hCLS1/mCLS1 was analyzed by Western blot using a V5-epitope A major protein of was found for tagged hCLS1/mCLS1 (Fig. the of hCLS1/mCLS1 the be which is consistent with their To this expression of the tagged hCLS1/mCLS1 was in Sf9 cells. to the in vitro translation a protein at was detected in the cell lysates by Western blot analysis (Fig. Thus, both hCLS1 and mCLS1 be expressed our experimental CLS enzyme activity in vitro and in cultured cells was In the reticulocyte hCLS1 expression increased CLS activity by control (Fig. Furthermore, recombinant hCLS1 was expressed in Sf9 insect cells, mitochondrial membrane CLS activity was found to be than that in membranes from (Fig. In addition, analysis of phospholipid from these cells an increase in CL of ± ± two no increase was seen with other phospholipids not The that CLS activity was found in expression DNA reticulocyte lysates the that the hCLS1 gene encodes a CLS not a of a CLS the 80.7% alignment with the conserved domain of PGP synthase, PGP synthase activity was also measured in the membrane fractions of Sf9 cells hCLS1. PGP synthase activity was not increased in cells hCLS1 ± ± in the of hCLS1 for the biosynthesis of CL. is well that CL biosynthesis occurs on the matrix side of the inner mitochondrial membrane via the CDP-DG pathway (17Schlame M. Haldar D. Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria.J. Biol. Chem. 1993; 268: 74-79Google Scholar). In the final step, CLS the of a from CDP-DG to PG to form CL. Although a rat CLS enzyme has been purified to homogeneity (18Schlame M. Hostetler K.Y. Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement.J. Biol. Chem. 1991; 266: 22398-22403Google Scholar), a gene encoding the enzyme has not been identified. In this study, we identified a cDNA that encodes the first mammalian CLS termed in humans and mice. that CLS1 is a CLS enzyme is by the First, expression of hCLS1 cDNA in reticulocyte lysates resulted in an increase in radiolabeled CL product (Fig. expression of hCLS1 cDNA in Sf9 cells resulted in a significant increase in CLS activities. In addition, this increase in CLS activities resulted in an increase in CL pool size in hCLS1-overexpressing cells. the expressed hCLS1 for CL synthesis, because no significant increase in PGP synthase activities was observed. high levels of hCLS1 transcripts were found in tissues (Fig. that have both high mitochondrial content and CLS activities (4Schlame M. Rua D. Greenberg M.L. The biosynthesis and functional role of cardiolipin.Prog. Lipid Res. 2000; 39: 257-288Google Scholar). CLS1 exhibits a in its domain structure and properties. All eukaryotic CLS proteins are membrane proteins with an domain an N-terminal mitochondrial target sequence by two membrane domains and three transmembrane the CDP-OH-P motif that is required for CLS activity also in the membrane regions (Fig. This topology a in which CL is synthesized in the matrix of the inner membrane near the (17Schlame M. Haldar D. Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria.J. Biol. Chem. 1993; 268: 74-79Google Scholar). conserved a high and a of In to these the hCLS1 was expressed in and tissues such as heart, skeletal muscle, and These data suggest that the cloned mammalian CLS1 is an conserved enzyme that is highly expressed in In we have identified and a gene encoding the first mammalian CLS in rat, and Because CLS1 the terminal for CL the for CL in mitochondrial function at the now be regulation and expression of CLS1 be used to define the role of CL in the of the hCLS1 gene has a for to whether CLS is a target for CL human such as Barth This was by a from the of Research is a Research in Cardiolipin

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