Comparative analyses of isoforms of the calcium-independent phosphatidylethanolamine N-acyltransferase PLAAT-1 in humans and mice
2016; Elsevier BV; Volume: 57; Issue: 11 Linguagem: Inglês
10.1194/jlr.m071290
ISSN1539-7262
AutoresZahir Hussain, Toru Uyama, Katsuhisa Kawai, Iffat Rahman, Kazuhito Tsuboi, Nobukazu Araki, Natsuo Ueda,
Tópico(s)Lipid metabolism and biosynthesis
ResumoN-Acylphosphatidylethanolamines (NAPEs) are a class of glycerophospholipids, which are known as precursors for different bioactive N-acylethanolamines. We previously reported that phospholipase A/acyltransferase-1 (PLAAT-1), which was originally found in mammals as a tumor suppressor, catalyzes N-acylation of phosphatidylethanolamines to form NAPEs. However, recent online database suggested the presence of an uncharacterized isoform of PLAAT-1 with an extra sequence at the N terminus. In the present study, we examined the occurrence, intracellular localization, and catalytic properties of this longer isoform, as well as the original shorter isoform from humans and mice. Our results showed that human tissues express the longer isoform but not the short isoform at all. In contrast, mice expressed both isoforms with different tissue distribution. Unlike the cytoplasmic localization of the shorter isoform, the long isoform was found in both cytoplasm and nucleus, inferring that the extra sequence harbors a nuclear localization signal. As assayed with purified proteins, neither isoform required calcium for full activity. Moreover, the overexpression of each isoform remarkably increased cellular NAPE levels. These results conclude that the new long isoform of PLAAT-1 is a calcium-independent N-acyltransferase existing in both cytoplasm and nucleus and suggest a possible formation of NAPEs in various membrane structures including nuclear membrane. J. Lipid Res. 2016. 57: 2051–2060. N-Acylphosphatidylethanolamines (NAPEs) are a class of glycerophospholipids, which are known as precursors for different bioactive N-acylethanolamines. We previously reported that phospholipase A/acyltransferase-1 (PLAAT-1), which was originally found in mammals as a tumor suppressor, catalyzes N-acylation of phosphatidylethanolamines to form NAPEs. However, recent online database suggested the presence of an uncharacterized isoform of PLAAT-1 with an extra sequence at the N terminus. In the present study, we examined the occurrence, intracellular localization, and catalytic properties of this longer isoform, as well as the original shorter isoform from humans and mice. Our results showed that human tissues express the longer isoform but not the short isoform at all. In contrast, mice expressed both isoforms with different tissue distribution. Unlike the cytoplasmic localization of the shorter isoform, the long isoform was found in both cytoplasm and nucleus, inferring that the extra sequence harbors a nuclear localization signal. As assayed with purified proteins, neither isoform required calcium for full activity. Moreover, the overexpression of each isoform remarkably increased cellular NAPE levels. These results conclude that the new long isoform of PLAAT-1 is a calcium-independent N-acyltransferase existing in both cytoplasm and nucleus and suggest a possible formation of NAPEs in various membrane structures including nuclear membrane. J. Lipid Res. 2016. 57: 2051–2060. Fatty acyl ethanolamides [N-acylethanolamines (NAEs)] are a class of lipid mediators. Among them, arachidonoylethanolamide (anandamide) is known to be an endogenous ligand of cannabinoid receptors (namely, an endocannabinoid) (1.Pacher P. Bátkai S. Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy.Pharmacol. Rev. 2006; 58: 389-462Crossref PubMed Scopus (1622) Google Scholar). In addition, palmitoylethanolamide and oleoylethanolamide show biological activities such as anti-inflammation, analgesia, and appetite suppression via different receptors including PPAR-α (2.Pavón F.J. Serrano A. Romero-Cuevas M. Alonso M. Rodríguez De Fonseca F. Oleoylethanolamide: a new player in peripheral control of energy metabolism. Therapeutic implications.Drug Discov. Today Dis. Mech. 2010; 7: e175-e183Crossref Scopus (20) Google Scholar, 3.Hesselink J.M.K. Hekker T.A. Therapeutic utility of palmitoylethanolamide in the treatment of neuropathic pain associated with various pathological conditions: a case series.J. Pain Res. 2012; 5: 437-442Crossref PubMed Scopus (58) Google Scholar, 4.Piomelli D. Hohmann A.G. Seybold V. Hammock B.D. A lipid gate for the peripheral control of pain.J. Neurosci. 2014; 34: 15184-15191Crossref PubMed Scopus (44) Google Scholar, 5.DiPatrizio N.V. Piomelli D. Intestinal lipid-derived signals that sense dietary fat.J. Clin. Invest. 2015; 125: 891-898Crossref PubMed Scopus (65) Google Scholar). These NAEs are biosynthesized principally through a two-step pathway from membrane glycerophospholipids via N-acylphosphatidylethanolamines (NAPEs) (6.Ueda N. Tsuboi K. Uyama T. Metabolism of endocannabinoids and related N-acylethanolamines: Canonical and alternative pathways.FEBS J. 2013; 280: 1874-1894Crossref PubMed Scopus (177) Google Scholar). The first reaction is the transfer of an acyl chain from a glycerophospholipid molecule such as phosphatidylcholine (PC) to the amino group of phosphatidylethanolamine (PE), resulting in the formation of NAPE, and the enzyme responsible is known as PE N-acyltransferase. Very recently, cPLA2ɛ, a member of the cytosolic phospholipase A2 (PLA2) family (PLA2G4), has been identified as the calcium-stimulated N-acyltransferase capable of catalyzing this step (7.Ogura Y. Parsons W.H. Kamat S.S. Cravatt B.F. A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines.Nat. Chem. Biol. 2016; 12: 669-671Crossref PubMed Scopus (79) Google Scholar). However, a series of our recent studies revealed that five members of the HRAS-like suppressor (HRASLS) family, which were originally discovered as tumor suppressors, possess calcium-independent phospholipid-metabolizing activities including NAPE-forming N-acyltransferase and PLA1/2 activities (8.Jin X-H. Okamoto Y. Morishita J. Tsuboi K. Tonai T. Ueda N. Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners.J. Biol. Chem. 2007; 282: 3614-3623Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 9.Uyama T. Morishita J. Jin X-H. Okamoto Y. Tsuboi K. Ueda N. The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type.J. Lipid Res. 2009; 50: 685-693Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10.Uyama T. Jin X-H. Tsuboi K. Tonai T. Ueda N. Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes.Biochim. Biophys. Acta. 2009; 1791: 1114-1124Crossref PubMed Scopus (61) Google Scholar, 11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12.Uyama T. Ikematsu N. Inoue M. Shinohara N. Jin X-H. Tsuboi K. Tonai T. Tokumura A. Ueda N. Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family.J. Biol. Chem. 2012; 287: 31905-31919Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and we proposed to give HRASLS-1–5 the names phospholipase A/acyltransferase-1–5 (PLAAT-1–5), respectively (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Among the five members, PLAAT-1 particularly received our attention because of its relatively high PE N-acyltransferase activity over PLA1/2 activity in vitro and predominant expression in testis, skeletal muscle, brain, and heart of humans, mice, and rats, where NAPEs accumulate in response to ischemia and inflammation (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13.Uyama T. Inoue M. Okamoto Y. Shinohara N. Tai T. Tsuboi K. Inoue T. Tokumura A. Ueda N. Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation.Biochim. Biophys. Acta. 2013; 1831: 1690-1701Crossref PubMed Scopus (22) Google Scholar). cDNAs of PLAAT-1 (originally referred to as A-C1) were cloned from a mouse chondrogenic cell line (ATDC5) (accession number, AF163095) and human renal cell carcinoma-derived cells (RCC-K1) by Akiyama et al. (14.Akiyama H. Hiraki Y. Noda M. Shigeno C. Ito H. Nakamura T. Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.J. Biol. Chem. 1999; 274: 32192-32197Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and Ito et al. (15.Ito H. Akiyama H. Shigeno C. Nakamura T. Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS).Cytogenet. Cell Genet. 2001; 93: 36-39Crossref PubMed Scopus (23) Google Scholar), respectively. Based on their nucleotide sequences, the primary structures of human and mouse PLAAT-1 proteins were deduced to be 168 and 167 amino acids long, respectively (14.Akiyama H. Hiraki Y. Noda M. Shigeno C. Ito H. Nakamura T. Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.J. Biol. Chem. 1999; 274: 32192-32197Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 15.Ito H. Akiyama H. Shigeno C. Nakamura T. Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS).Cytogenet. Cell Genet. 2001; 93: 36-39Crossref PubMed Scopus (23) Google Scholar). We reported that the recombinant PLAAT-1 proteins with these primary structures show the aforementioned enzyme activity (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13.Uyama T. Inoue M. Okamoto Y. Shinohara N. Tai T. Tsuboi K. Inoue T. Tokumura A. Ueda N. Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation.Biochim. Biophys. Acta. 2013; 1831: 1690-1701Crossref PubMed Scopus (22) Google Scholar). However, recently the NCBI database exhibited the existence of a longer isoform with an extra amino acid sequence at the N terminus in various mammals, including humans (NM_020386) and mice (XM_006522203). The deduced amino acid sequence of this longer isoform comprised 273 (humans) or 277 (mice) amino acids, respectively. In this article, we will tentatively refer to the original shorter isoform and later reported longer isoform of humans (h) and mice (m) as hPLAAT-1S and hPLAAT-1L or mPLAAT-1S and mPLAAT-1L, respectively (Fig. 1). Moreover, the database suggested that mice have two different transcripts encoding mPLAAT-1S (NM_013751 and XM_006522204). We will refer to NM_013751 and XM_006522204 as mPLAAT-1SA and mPLAAT-1SB (Fig. 2), respectively.Fig. 2The structures of mRNAs encoding PLAAT-1 isoforms. The schematic organization of mRNAs of human (A) and mouse (B) PLAAT-1 isoforms is shown. "E" stands for an exon, and the lines between exons indicate introns. Asterisks and arrows indicate the positions of initiation codons and primer sets A–F, respectively. In B, 2′ and a gray line indicate part of exon 2 and part of the intron between exons 2 and 3, respectively, which are contained in mPLAAT-1SB. Accession numbers are shown in parentheses. Chr, chromosome.View Large Image Figure ViewerDownload Hi-res image Download (PPT) So far, endogenous PLAAT-1 has been examined without discriminating between PLAAT-1S and PLAAT-1L. Thus, the first aim of the present study was to clarify whether both isoforms are endogenously expressed in human and mouse tissues. Second, because we noticed that the N-terminal extra domain of PLAAT-1L is a kind of polybasic domain (see Results for details), we investigated the intracellular localization. Moreover, because only recombinant PLAAT-1S protein has been characterized, our third aim was to examine the catalytic properties of recombinant PLAAT-1L. The results suggest that PLAAT-1L is endogenously expressed in both human and mouse tissues, whereas PLAAT-1S exists only in mice. We also report that PLAAT-1L localizes in both nucleus and cytoplasm and functions as an NAPE-forming N-acyltransferase in a Ca2+-independent manner. 1,2-[1′-14C]dipalmitoyl-PC was purchased from PerkinElmer Life Science (Boston, MA). [1,2-14C]ethanolamine HCl was from Moravek Biochemicals (Brea, CA). Horseradish peroxidase-linked anti-mouse and anti-rabbit IgGs were from GE Healthcare (Piscataway, NJ). 1,2-Dipalmitoyl-PC, 1,2-dioleoyl-PE, mouse anti-FLAG monoclonal antibody M2, anti-FLAG M2-conjugated agarose affinity gel, and FLAG peptide were from Sigma-Aldrich (St. Louis, MO). Monoclonal antibodies against lamin A/C and syntaxin 6 and rabbit anti-FLAG (DYKDDDDK) polyclonal antibody were from Cell Signaling Technology (Danvers, MA). Dulbecco's modified Eagle's medium, fetal bovine serum, Lipofectamine 2000, TRIzol, pEF1/Myc-His vector, pEF6/Myc-His vector, and Alexa Fluor® 488-conjugated goat anti-mouse IgG were from Invitrogen/Thermo Fisher Scientific (Carlsbad, CA). Triton X-100, Nonidet P-40, and Proteinase K were from Nacalai Tesque (Kyoto, Japan). KOD-Plus-Neo and KOD FX DNA polymerases were from Toyobo (Osaka, Japan). Protein assay dye reagent concentrate was from Bio-Rad (Hercules, CA). Protease inhibitor cocktail set III, Immobilon-P, and precoated silica gel 60 F254 aluminum sheets (20 × 20 cm, 0.2 mm thick) for TLC were from Merck (Darmstadt, Germany). Human MTC™ Panels I and II were purchased from Clontech (Mountain View, CA). An NE-PER Nuclear and Cytoplasmic Extraction Reagents kit and Pierce® Western Blotting Substrate Plus were purchased from Thermo Fisher Scientific (Rockford, IL). Paraformaldehyde fixative solution was from Muto Pure Chemicals (Tokyo, Japan). Normal goat serum was from Vector Laboratories (Burlingame, CA). Permafluor was from Immunotech (Marseille, France). Silver Staining MS Kit was from Wako Pure Chemical Industries (Osaka, Japan). Ex Taq DNA polymerase, Prime-STAR DNA polymerase and a PrimeScript RT reagent kit were from TaKaRa Bio (Ohtsu, Japan). Human prostate cancer cell line (Du-145) was obtained from American Type Culture Collection (Rockville, MD). pEF6/Myc-His expression vectors harboring the cDNAs of the C-terminally FLAG-tagged PLAAT-1S from humans (hPLAAT-1S-FL) and mice (mPLAAT-1S-FL) were constructed as described previously (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The cDNA encoding the C-terminally FLAG-tagged hPLAAT-1L (hPLAAT-1L-FL) was obtained from human skeletal muscle cDNA in MTC™ Panel I by PCR amplification using two pairs of nested primers. The first PCR using primers F1 and R1 (Table 1) was carried out with KOD-Plus-Neo DNA polymerase for 30 cycles at 98°C for 10 s, 55°C for 30 s, and 68°C for 60 s, and the second PCR using primers F2 and R2 was for 25 cycles under the same conditions. The obtained DNA fragment was inserted between SpeI and NotI sites of the pEF1/Myc-His vector. The cDNA encoding the C-terminally FLAG-tagged mPLAAT-1L (mPLAAT-1L-FL) was constructed in two steps of PCR using primers F3 and R3. The first step PCR amplification was carried out with Prime-STAR DNA polymerase for 35 cycles at 98°C for 10 s, 61°C for 30 s, and 72°C for 60 s. cDNA used as a template was prepared from mouse heart as described in the next section. The second step PCR was carried out using the first PCR product as a template for 15 cycles at 98°C for 10 s, 55°C for 30 s, and 72°C for 60 s. The obtained DNA fragment was inserted between SpeI and NotI sites of the pEF6/Myc-His vector. All constructs were sequenced in both directions using an Applied Biosystems 3130 Genetic Analyzer (Thermo Fisher Scientific).TABLE 1Primers used for the construction of expression vectorscDNA (Accession Number)PrimeraF, forward primer; R, reverse primer.SequencebRestriction sites and FLAG tag sequences are indicated as small letters.Location of NucleotideshPLAAT-1L(NM_020386)F15′-CCAAGCGAGGTCTAGCCGGAGCGACTGTGC-3′31–60R15′-TCCTCCCAAATTCCTTCAATATCAATATC-3′956–928F25′-cgcactagtccaagATGGTCAGAGCCTCGTGCCGGCTCGGC-3′ (SpeI site)92–118R25′-cgcgcggccgcctacttatcgtcgtcatccttgtaatcATAGTATTTTGCTCTTTGTCCTTTTGGAAAC-3′ (NotI site followed by an in-frame FLAG sequence)910–880mPLAAT-1L(XM_006522203)F35′-cgcactagtccaagATGCCTGAGGCGTGCTTGGAGGATTTG-3′ (SpeI site)16–42R35′-cgcgcggccgcctacttatcgtcgtcatccttgtaatcATATTTCGTTCTTTGTCTTTTGGG-3′ (NotI site followed by an in-frame FLAG sequence)846–823a F, forward primer; R, reverse primer.b Restriction sites and FLAG tag sequences are indicated as small letters. Open table in a new tab To see the tissue distribution of hPLAAT-1 isoforms, cDNAs from human MTC™ Panels I and II were used as templates and subjected to PCR amplification by Ex Taq DNA polymerase. As shown in Table 2 and Fig. 2, the primers were designed on the basis of a common sequence to hPLAAT-1S and hPLAAT-1L [primer set A, which was used previously (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar)], a unique sequence for hPLAAT-1L (primer set B), and 5′-untranslated region (5′-UTR) of hPLAAT-1S (primer set C). The PCR conditions used were as follows: 30 cycles with denaturation at 94°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s for primer set A; 35 cycles with denaturation at 98°C for 10 s, annealing and extension at 68°C for 60 s for primer set B; 30 cycles with denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 68°C for 60 s for primer set C; 25 cycles with denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 68°C for 60 s for human GAPDH (hGAPDH) as a control. Du-145 cell line was chosen arbitrarily to prepare human genomic DNA. The cells were homogenized in 100 mM Tris-HCl (pH 8.5) containing 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and Proteinase K. After centrifugation for 5 min at 20,400 g, the genomic DNA was precipitated by adding isopropanol, washed with 70% ethanol, and resuspended in 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA.TABLE 2Primers used for PCRcDNA (Accession Number)Primer SetDirectionSequenceLocation of NucleotideshPLAAT-1L (NM_020386) and hPLAAT-1SAForward5′-CCCTGTGGAAGAAATCATAAAGCGGTC-3′685–711Reverse5′-CCCAGGAATGAGAAGACACCAACAGC-3′876–851hPLAAT-1L (NM_020386)BForward5′-TGCCTCCCGGTGCGAGAAGAAGACC-3′366–390Reverse5′-ACAGGGCCCAGTGCTGATAGCCAGG-3′506–4825′-UTR of hPLAAT-1S [reported by Ito et al. (15.Ito H. Akiyama H. Shigeno C. Nakamura T. Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS).Cytogenet. Cell Genet. 2001; 93: 36-39Crossref PubMed Scopus (23) Google Scholar)]CForward5′-CCCCTTGTGCACACATCAGTGTTGG-3′220–244Reverse5′-AAGAAGAGCTAGCTAATACAACTTGCC-3′384–358hGAPDH (NM_002046)—Forward5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′199–224Reverse5′-CATGTGGGCCATGAGGTCCACCAC-3′1,181–1,158mPLAAT-1L (XM_006522203)DForward5′-TGCCTGAGGCGTGCTTGGAGGATTTG-3′17–42Reverse5′-CGCCGCGCCACCTCTGATCCCTCCGC-3′166–141mPLAAT-1SA (NM_013751)EForward5′-CTGAGCTGTGAGCAGGCGATTTGTGTG-3′39–65Reverse5′-AGCCATCACCCAAGTACAGTGCCCAG-3′230–205mPLAAT-1SB (XM_006522204)FForward5′-TAAGACAACCCAGCTTGAGCAGGGAG-3′177–202Reverse5′-AGCCATCACCCAAGTACAGTGCCCAG-3′317–292mGAPDH (NM_008084)—Forward5′-AACTCCCACTCTTCCACCTTCGATG-3′1,099–1,123Reverse5′-CCTGTTGCTGTAGCCGTATTCATTG-3′1,203–1,179 Open table in a new tab To see the tissue distribution of mPLAAT-1 isoforms, C57BL/6 mice (male, 8 weeks old; Japan SLC Inc.) were anesthetized and euthanized by decapitation according to the guidelines for care and use of animals established by Kagawa University (Kagawa, Japan). Total RNAs were then isolated from different mouse tissues using TRIzol. First strand cDNAs were prepared from 5 µg of total RNA using a PrimeScript RT reagent kit and subjected to PCR amplification by KOD FX DNA polymerase. Unique sequences for mPLAAT-1L (primer set D), mPLAAT-1SA (primer set E) and mPLAAT-1SB (primer set F) were used to design primers (Table 2 and Fig. 2). The PCR conditions used were as follows: 35 cycles with denaturation at 98°C for 10 s, annealing and extension at 72°C for 60 s for primer sets D–F; 28 cycles with denaturation at 98°C for 10 s, annealing at 58°C for 30 s, and extension at 68°C for 60 s for mouse GAPDH (mGAPDH) as a control. COS-7 cells were grown at 37°C to 90% confluency in 100 mm plastic dishes containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a humidified 5% CO2 and 95% air incubator. The expression vectors harboring cDNAs for FLAG-tagged PLAAT-1 isoforms were introduced into COS-7 cells using Lipofectamine 2000 according to the manufacturer's instructions. Forty-eight hours after transfection, cells were harvested and sonicated twice each for 5 s in 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl. As described previously for the purification of PLAAT-1S (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), the cell homogenates overexpressing human PLAAT-1 isoforms were subjected to ultracentrifugation at 105,000 g for 30 min, and PLAAT-1 proteins were purified from the resultant supernatant (cytosol) by anti-FLAG antibody affinity chromatography. The eluted fractions were analyzed by silver staining. The protein concentration was determined by the method of Bradford with BSA as a standard (16.Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215507) Google Scholar). COS-7 cells transiently expressing FLAG-tagged PLAAT-1 isoforms were harvested after 48 h of transfection, suspended in 20 mM Tris-HCl (pH 7.4) containing 0.25 M sucrose and 1 mM EDTA, and then homogenized by sonic disruption twice for 5 s each. Each sample (20–30 µg of protein) was separated by SDS-PAGE on 14% gel and electrotransferred to a hydrophobic polyvinylidene difluoride membrane (Immobilon-P). The membrane was blocked with PBS containing 5% dried skimmed milk and 0.1% Tween 20 (buffer A) and then incubated with anti-FLAG antibody (1:2,000 dilution) in buffer A at room temperature for 1 h, followed by incubation with horseradish peroxidase-labeled anti-rabbit IgG antibody (1:4,000 dilution) in buffer A at room temperature for 1 h. The membrane was finally treated with Pierce® Western Blotting Substrate Plus and visualized with the aid of a LAS1000plus lumino-imaging analyzer (FUJIX Ltd., Japan). COS-7 cells overexpressing FLAG-tagged PLAAT-1 isoforms were cultured on 18 mm glass coverslips containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C for 24 h after transfection. The cells were then fixed with 4% paraformaldehyde in 0.1 M PBS for 15 min. The fixed cells were rinsed with PBS twice and permeabilized with 0.2% Triton X-100 in PBS for 15 min. After blocking with 10% normal goat serum in PBS for 1 h, the cells were incubated with anti-FLAG antibody (1:500 dilution) in 1% normal goat serum in PBS for 1 h at room temperature. The cells were washed with PBS twice and labeled with Alexa 488-conjugated anti-mouse IgG (1:1,000 dilution) in 1% normal goat serum in PBS for 1 h in the dark. The cells were washed with PBS twice, and the specimen coverslips were mounted on glass slides using Permafluor, a mounting medium, and were observed with an LSM 700 confocal laser microscope (Carl Zeiss, Germany). FLAG-tagged PLAAT-1 isoforms were expressed in COS-7 cells as described in Expression and Purification of Recombinant Proteins. After 48 h of transfection, the nuclear-cytoplasmic fractionation was conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit according to the manufacturer's protocol (17.Tsai N-P. Lin Y-L. Tsui Y-C. Wei L-N. Dual action of epidermal growth factor: extracellular signal-stimulated nuclear-cytoplasmic export and coordinated translation of selected messenger RNA.J. Cell Biol. 2010; 188: 325-333Crossref PubMed Scopus (29) Google Scholar). The obtained nuclear and postnuclear supernatant fractions were then subjected to Western blotting as described above, using antibodies against FLAG (1:2,000 dilution), lamin A/C (1:2,000 dilution), and syntaxin 6 (1:2,000 dilution) as primary antibodies and horseradish peroxidase-labeled anti-mouse and anti-rabbit IgGs (1:4,000 dilution) as secondary antibodies. Purified human PLAAT-1 isoforms (0.2 µg of protein) were incubated with 40 µM 1,2-[1′-14C]dipalmitoyl-PC (45,000 cpm) and 80 µM 1,2-dioleoyl-PE in 100 µl of 100 mM glycine-NaOH buffer (pH 8.2), 2 mM dithiothreitol, and 0.1% Nonidet P-40 at 37°C for 30 min. In some assays, 1 mM EDTA or 1 mM CaCl2 was also added. Reactions were terminated by the addition of 320 µl of a mixture of chloroform and methanol (2:1, v/v) containing 5 mM 3(2)-t-butyl-4-hydroxyanisole. After centrifugation, 100 µl of the organic phase was spotted on a silica gel thin-layer plate (10 cm height) with a calibrated capillary glass pipet connected to a rubber aspirator tube (Drummond Scientific Co., Broomall, PA) and was dried under the airflow of a hair dryer. Later on, the TLC plate was developed at 4°C for 25 min in a mixture of chloroform/methanol/28% ammonium hydroxide (80:20:2, v/v) (solvent A). The distribution of radioactivity on the plate was visualized and quantified using an image reader FLA-7000 (Fujifilm, Tokyo, Japan). COS-7 cells transiently expressing PLAAT-1 isoforms were grown at 37°C to 80% confluency in 6-well plates containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and were labeled with [14C]ethanolamine (0.32 µCi/well) for 18 h. Cells were then harvested and washed twice with PBS. Total lipids were extracted by the method of Bligh and Dyer (18.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42648) Google Scholar), spotted on a silica gel thin-layer plate (20 cm height), and developed at 4°C for 90 min in solvent A. The distribution of radioactivity on the plate was visualized and quantified using an image reader FLA-7000. We previously cloned cDNAs of PLAAT-1S from human testis and mouse brain (11.Shinohara N. Uyama T. Jin X-H. Tsuboi K. Tonai T. Houchi H. Ueda N. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.J. Lipid Res. 2011; 52: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In the present study, we cloned cDNAs of PLAAT-1L from human skeletal muscle and mouse heart based on the reported nucleotide sequences (accession number NM_020386 and XM_006522203, respectively). The sequences of cDNAs that we cloned were fully coincident with the reported ones. These results showed that human skeletal muscle and mouse heart express mRNA of PLAAT-1L. Comparing with PLAAT-1S, the deduced amino acid sequences of human and mouse PLAAT-1L had an N-terminal extra sequence comprising 105 and 110 amino acids, respectively (Fig. 1). The sequences of PLAAT-1L except the extra sequences were exactly identical with those of PLAAT-1S in both humans and mice. Though mPLAAT-1S showed 83.9% amino acid identity with hPLAAT-1S, the identity between the extra sequences of hPLAAT-1L and mPLAAT-1L was as low as 18.1%. However, as pointed by asterisks in Fig. 1, both the extra sequences were abundant in basic amino acids (20 Arg and 1 His in hPLAAT-1L and 17 Arg, 2 Lys, and 2 His in mPLAAT-1L), and their isoelectric points were 12.09 and 11.50, respectively. Thus, the extra sequence of PLAAT-1L appeared to form a polybasic domain. As shown in Fig. 2A, hPLAAT-1L mRNA comprises four exons (exons 1–4) (NM_020386), while hPLAAT-1S mRNA lacks exon 1 (15.Ito H. Akiyama H. Shigeno C. Nakamura T. Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS).Cytogenet. Cell Genet. 2001; 93: 36-39Crossref PubMed Scopus (23) Google Scholar). We found that most of the 5′-UTR sequence of hPLAAT-1S mRNA [36–401 in (15.Ito H. Akiyama H. Shigeno C. Nakamura T. Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS).Cytogenet. Cell Genet. 2001; 93: 36-39Crossref PubMed Scopus (23) Google Scholar)] is identical to the 3′ sequence of the intron between exon 1 and 2 and ended b
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