Cloning, Expression, and Characterization of a Human 4′-Phosphopantetheinyl Transferase with Broad Substrate Specificity
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m305459200
ISSN1083-351X
AutoresAnil K. Joshi, Lei Zhang, Vangipuram S. Rangan, Sean C. Smith,
Tópico(s)Biochemical and Molecular Research
ResumoA single candidate 4′-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4′-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4′-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the α-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336–342). Thus, in contrast to yeast, which utilizes separate 4′-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4′-phosphopantetheinylation reactions. A single candidate 4′-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4′-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4′-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the α-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336–342). Thus, in contrast to yeast, which utilizes separate 4′-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4′-phosphopantetheinylation reactions. The fatty acid synthases (FASs) 1The abbreviations used are: FAS, fatty acid synthase; PKS, polyketide synthase; ACP, acyl carrier protein; ACPmit, mitochondrial acyl carrier protein; ACPfas, acyl carrier protein domain of the fatty acid synthase; PPTase, 4′-phosphopantetheine transferase; Sfp, 4′-phoshopantetheine transferase involved in surfactin production in B. subtilis; PCP, peptidyl carrier protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; HPLC, high performance liquid chromatography; EST, expressed sequence tag; Ni-NTA, nickel-nitrilotriacetic acid; TEV, tobacco etch virus. associated with the soluble cytoplasm of yeast and animal cells comprise large multifunctional polypeptides that contain all of the catalytic components required for the synthesis of long-chain fatty acids from malonyl-CoA de novo. These multifunctional polypeptides are commonly referred to as type I FASs. The animal FASs consist of two identical polypeptides of approximately 2500 residues (α2), whereas the yeast FAS comprises six copies each of two nonidentical polypeptides (α6β6; α = 1845, β = 1887 residues) (1Smith S. Witkowski A. Joshi A.K. Prog. Lipid Res. 2003; 42: 289-317Crossref PubMed Scopus (498) Google Scholar, 2Kolodziej S.J. Penczek P.A. Schroeter J.P. Stoops J.K. J. Biol. Chem. 1996; 271: 28422-28429Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In most bacteria and in plant plastids, the various catalytic components of the FAS are present on separate discrete polypeptides and are commonly referred to as type II FASs (3Heath R. Jackowski S. Rock C. Vance D. Je V. Biochemistry of Lipids, Lipoproteins and Membranes. 4th Ed. Elsevier, Amsterdam2002: 55-92Google Scholar). An essential component of both type I and type II systems is a small molecular mass domain/polypeptide known as an acyl carrier protein (ACP) that is posttranslationally modified, by insertion of a 20-Å-long phosphopantetheinyl moiety, derived from CoA, to a positionally conserved serine residue (4Lambalot R.H. Gehring A.M. Flugel R.S. Zuber P. LaCelle M. Marahiel M.A. Reid R. Khosla C. Walsh C.T. Chem. Biol. 1996; 3: 923-936Abstract Full Text PDF PubMed Scopus (725) Google Scholar). The terminal sulfhydryl of the phosphopantetheinyl moiety provides the site of covalent attachment of the substrates and the growing fatty acyl chain, so that the phosphopantetheine plays an essential role as a "swinging arm" in the translocation of intermediates between different catalytic sites of the FASs (5Lynen F. Eur. J. Biochem. 1980; 112: 431-442Crossref PubMed Scopus (131) Google Scholar). ACPs fulfill a similar role in the type I and type II polyketide synthases (PKSs) found mainly in bacteria and fungi that are capable of elaborating a broad range of secondary metabolites. Fungi (6Brody S. Mikolajczyk S. Eur. J. Biochem. 1988; 173: 353-359Crossref PubMed Scopus (54) Google Scholar, 7Schneider R. Massow M. Lisowsky T. Weiss H. Curr. Genet. 1995; 29: 10-17Crossref PubMed Scopus (93) Google Scholar), animal, and plant cells also contain a type II FAS system in their mitochondria. The role of the mitochondrial FASs is not well established, but it has been suggested that, at least in fungi and plants, they may serve to provide octanoate, the precursor of lipoic acid and/or long-chain fatty acids that are used in the remodeling of mitochondrial phospholipids (7Schneider R. Massow M. Lisowsky T. Weiss H. Curr. Genet. 1995; 29: 10-17Crossref PubMed Scopus (93) Google Scholar, 8Wada H. Shintani D. Ohlrogge J. Proc. Natl. Acad. Sci. USA. 1997; 94: 1591-1596Crossref PubMed Scopus (176) Google Scholar, 9Gueguen V. Macherel D. Jaquinod M. Douce R. Bourguignon J. J. Biol. Chem. 2000; 275: 5016-5025Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 10Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (133) Google Scholar). The ACP component of the mitochondrial FAS appears to be associated with the respiratory complex I in animals and in Neurospora crassa (11Runswick M.J. Fearnley I.M. Skehel J.M. Walker J.E. FEBS Lett. 1991; 286: 121-124Crossref PubMed Scopus (158) Google Scholar, 12Sackmann U. Zensen R. Rohlen D. Jahnke U. Weiss H. Eur. J. Biochem. 1991; 200: 463-469Crossref PubMed Scopus (117) Google Scholar). Phosphopantetheinylated carrier proteins also play an essential role as components of the non-ribosomal peptide synthases found in microorganisms that are responsible for producing a variety of short peptides containing both proteogenic and unusual amino acids (13Weber T. Marahiel M.A. Structure. 2001; 9: R3-R9Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The nonribosomal peptide synthases also comprise multifunctional polypeptides in which the role of the carrier protein domain is to translocate amino acyl moieties from an adenylation domain to a condensation domain, where formation of a new peptide bond takes place (14Cane D.E. Walsh C.T. Khosla C. Science. 1998; 282: 63-68Crossref PubMed Scopus (561) Google Scholar). Enzymes capable of phosphopantetheinylating carrier proteins involved in the biosynthesis of fatty acids, polyketides, and peptides have been identified and characterized from a variety of sources. Many organisms have more than one phosphopantetheine transferase (PPTase), and different PPTases commonly are utilized to service carrier proteins associated with FASs and non-ribosomal peptide synthases within the same species (4Lambalot R.H. Gehring A.M. Flugel R.S. Zuber P. LaCelle M. Marahiel M.A. Reid R. Khosla C. Walsh C.T. Chem. Biol. 1996; 3: 923-936Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 15Mootz H.D. Finking R. Marahiel M.A. J. Biol. Chem. 2001; 276: 37289-37298Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Yeast utilizes separate PPTases for phosphopantetheinylation of the ACP domain of the cytosolic type I FAS and the mitochondrial type II ACP. Remarkably, the former is actually a constituent domain of the α-subunit of the type I cytosolic FAS, so that this protein is capable of self-phosphopantetheinylation (16Fichtlscherer F. Wellein C. Mittag M. Schweizer E. Eur. J. Biochem. 2000; 267: 2666-2671Crossref PubMed Scopus (85) Google Scholar), whereas the latter is a separate PPTase that acts only on the mitochondrial ACP (17Stuible H.P. Meier S. Wagner C. Hannappel E. Schweizer E. J. Biol. Chem. 1998; 273: 22334-22339Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). PPTases have been classified into three groups, based on sequence and structural similarity and substrate specificity (15Mootz H.D. Finking R. Marahiel M.A. J. Biol. Chem. 2001; 276: 37289-37298Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Members of the first group, typified by the AcpS of Esch-erichia coli,are characteristically ∼120 residues long, function as homotrimers, and have fairly narrow substrate specificities limited to the ACPs of type II FAS and PKS systems (18Suo Z. Tseng C.C. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 99-104Crossref PubMed Scopus (34) Google Scholar). Members of the second group, typified by the Sfp of Bacillus subtilis, are usually at least 240 residues long, function as monomers, and have very broad substrate specificities that include carrier proteins associated with non-ribosomal peptide synthases, FASs, and PKSs (18Suo Z. Tseng C.C. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 99-104Crossref PubMed Scopus (34) Google Scholar, 19Quadri L.E. Weinreb P.H. Lei M. Nakano M.M. Zuber P. Walsh C.T. Biochemistry. 1998; 37: 1585-1595Crossref PubMed Scopus (541) Google Scholar). Membership in the third group is reserved for those PPTases that are attached covalently to type I FASs, such as that associated with the yeast cytosolic FAS (16Fichtlscherer F. Wellein C. Mittag M. Schweizer E. Eur. J. Biochem. 2000; 267: 2666-2671Crossref PubMed Scopus (85) Google Scholar). The PPTases involved in phosphopantetheinylation of the ACPs involved in fatty acid synthesis in animals have not yet been identified. Thus, the objective of this study was to identify the PPTase(s) responsible for posttranslational modification of the ACP domain of the type I cytosolic FAS and the ACP associated with the type II mitochondrial FAS in humans. Cloning, Expression, and Purification of Human PPTase—Based on the available sequence information (accession nos. GI 7106836, GI 9295190, and EST cDNA ATCC 1849005), PCR primers (Table I) were designed to amplify full-length (∼1 kb) putative human PPTase cDNA from human liver and brain cDNA libraries (Clontech, Palo Alto, CA). The PCR procedure employed was essentially as described previously (20Joshi A.K. Smith S. J. Biol. Chem. 1993; 268: 22508-22513Abstract Full Text PDF PubMed Google Scholar). To improve the specificity of the PCR, two sets of primers were used to amplify cDNA. The first round of PCR, with primer pair T4/B3 and template DNA from the two human cDNA libraries, resulted in the amplification of an expected sized (∼1 kb) product, indicating the presence of PPTase transcripts in both the libraries. The amplified DNA from the first round PCR reaction was then used as template for the second round, using a nested PPTase-specific primer pair T2/B1. Again, an amplified product of ∼1 kb was obtained using DNA template arising from both human cDNA libraries. The amplified fragment from the two sources was purified, using QIAquick PCR purification kit (Qiagen Inc., Valencia, CA), digested at KpnI and PstI sites that had been engineered into the amplification primers T2 and B1, respectively, and cloned into the E. coli expression vector pQE80 L (Qiagen). To facilitate purification of the encoded proteins by metal ion affinity chromatography, a cleavable His6 tag was engineered at the start of the cloned cDNAs. Authenticity of the cloned PCR fragments was confirmed by DNA sequencing using vector-specific primers. The sequences of the amplified cDNAs from the two libraries were found to be identical. The amino acid sequence of the encoded protein is shown in Fig. 1. Protein expression from the cloned cDNA was attempted in E. coli DH5α and BL21-codon plus strains; however, most of the expressed PPTase was found to be present in inclusion bodies.Table IPCR primers used in this studyPrimersSequenceLocationaThe base pair numbers for PPTase T9 correspond to Tev. T linker; numbers for other PPTase-specific primers are according PPTase sequence in accession no. GI 7106836, for ACPfas specific primers base pairs are according to GI 915391, while for ACPmit the numbers are according EST cDNA clone IMAGE-5287610.PPTasePPTase T25′-atcatggtaccATGGTTTTCCCTGCCAAACGGTTCT167-191 bpKpnIPPTase T45′-ATAGCGGCGAGGTCCGCTTTCAGT141-164 bpPPTase B15′-tatagctgcagTCATGACTTTGTACCATTTCGTAT1073-1096 bpPstIPPTase B35′-ATTTCCCTTTGTTACTCAGGGAATCAT1096-1122 bpPPTase T95′-catcacgcgcgCGATTACGATATCCCAACGACCGA5-28 bpBssHIIPPTase B55′-gcttgcggccgcTCATGACTTTGTACCATTTCGTA1074-1096 bpNotIACPfasACPfas T5′-tcatggtaccAGGGACAGGGACAGCCAGCGGGA6472-6494 bpKpnIACPfas B5′-tataaagct tcaCTCCTTGGGCGTGGGGCATGCCA6716-6738 bpHindIIIACPmitACPmit T15′-atcatggtaccATGGCGTCTCGTGTCCTTTCA15-35 bpKpnIACPmit B15′-tatagctgcagTTATTCATATACATCCTTCTTATCTG460-485 bpPstIACPmit T25′-ctattctagagccATGGCGTCTCGTGTCCTTTCAGCCTAT15-41 bpXbaIACPmit T35′-cgattctagagccatgTATAGCGACATGCCTCCTTTGA216-237 bpXbaIACPmit B25′-tcatagctagcTTCATATACATCCTTCTTATCT461-482 bpNheITEV linkerTev. T5′-gatccgattacgatatcccaacgaccgaaaacctgtattttcagggtacBamHI KpnITev. B5′-cctgaaaatacaggttttcggtcgttgggatatcgtaatcga The base pair numbers for PPTase T9 correspond to Tev. T linker; numbers for other PPTase-specific primers are according PPTase sequence in accession no. GI 7106836, for ACPfas specific primers base pairs are according to GI 915391, while for ACPmit the numbers are according EST cDNA clone IMAGE-5287610. Open table in a new tab To overcome this problem, the cDNA encoding the putative human PPTase in pQE80 L was reamplified using PPTase primers T9 and B5 and cloned into a modified baculoviral transfer vector, pFast Bac 1 (Invitrogen, Carlsbad, CA), using appropriate restriction sites engineered into the two primers. Authenticity of the cDNA sequence was confirmed, recombinant baculoviral stocks were generated, and the encoded protein was expressed in Sf9 insect cells, using the Bac-To-Bac baculoviral expression system (Invitrogen), according to the instructions from the manufacturer. The infected Sf9 cells were homogenized at 4 °C in buffer A (250 mm potassium phosphate buffer, pH 7, containing 10% glycerol) and centrifuged at 4 °C, 144,000 × g for 60 min. The supernatant was filtered (0.45 μm) and loaded at 20 °C onto a 5-ml HiTrap Chelating HP column (Amersham Biosciences). The column was washed with 50 ml of buffer A and 40 ml of buffer B (60 mm imidazole in buffer A), and bound proteins were eluted with a 100-ml gradient of 60–400 mm imidazole in buffer A. Fractions containing the enzyme were pooled and dialyzed at 4 °C against buffer C (50 mm potassium phosphate buffer, pH 7, containing 10% glycerol and 1 mm dithiothreitol). Recombinant PPTase was further purified at 20 °C by HPLC using a column of DE-53 anion exchanger equilibrated with buffer C. The unbound protein fraction, containing PPTase activity, was collected and concentrated at 4 °C using a Vivaspin concentrator (Vivascience AG, Hannover, Germany). The purified PPTase protein was stored at –70 °C, typically at a concentration of 1 mg/ml. Cloning and Expression of the ACP Domain of the Human Cytosolic FAS (ACPfas)—Appropriate primers (Table I) were used to amplify a fragment, from human liver and brain cDNA libraries, corresponding to residues 2117–2205 of the human FAS. To facilitate purification of the encoded protein by nickel ion affinity chromatography, a cleavable N-terminal His6 tag was encoded in the ACPfas. The amplified fragments from the two sources were purified using QIAquick PCR purification kit (Qiagen), digested at the KpnI and HindIII sites that had been engineered into the amplification primers ACPfas T and ACPfas B, respectively, and cloned into the E. coli expression vector pQE80 L (Qiagen), using standard ligation and transformation procedures. Authenticity of the cloned PCR fragments from the two libraries was confirmed by DNA sequencing, using vector-specific primers. The clones derived from liver and brain libraries had identical sequences. The encoded amino acid sequence is shown in Fig. 1. The recombinant plasmid was expressed in the E. coli BL21-codon plus strain. Cells were grown at 37 °C in LB medium to A 600 of 0.5–0.7 and induced with 1 mm IPTG for 3 h. Cells were suspended in 0.25 m potassium phosphate buffer, pH 7, containing 10% glycerol and protease inhibitors, and lysed using a French pressure cell press. The lysate was centrifuged at 50,000 × g for 1 h at 4 °C. The recombinant ACP was purified from the soluble extract of E. coli cells, by a combination of nickel ion affinity chromatography and gel filtration. The soluble extract was applied to a nickel-nitrilotriacetic acid column equilibrated with 0.25 m potassium phosphate buffer, pH 7, and 10% glycerol. The column was then washed sequentially with the equilibration buffer containing 50, 100, and 250 mm imidazole. Fractions containing ACPfas (eluting at 100 mm imidazole) were pooled, concentrated using a Vivaspin concentrator, and chromatographed on a column of Biogel P30 (40 × 1.4 cm column eluted with 0.1 m potassium phosphate buffer, pH 7, at 13 ml/h, 20 °C). Cloning, Expression, and Purification of the Human Mitochondrial ACP (ACPmit)—Based on BLAST searches of the human genome sequence data base, using authentic type I and type II ACPs, we identified a putative ACPmit sequence that was represented in two human EST clones (IMAGE clones 4799845 and 4816243). Sequence alignments with authentic type II ACP sequences indicated that these ESTs likely encoded a full-length human ACPmit, together with an N-terminal extension of ∼67 residues that could constitute a mitochondrial targeting sequence. Specific primers, ACPmit T1/B1 (Table I) with appropriate restriction sites, were designed, and the cDNA encoding the putative ACPmit was amplified using the PCR, essentially as described previously (20Joshi A.K. Smith S. J. Biol. Chem. 1993; 268: 22508-22513Abstract Full Text PDF PubMed Google Scholar). The amplified fragments from the two sources were purified using a QIAquick PCR purification kit (Qiagen), then cleaved at the introduced KpnI and PstI sites and cloned into the E. coli expression vector pQE80 L (Qiagen). To facilitate purification of the encoded protein by metal-ion affinity chromatography, a cleavable His6 tag was encoded at the start of the cDNA. Authenticity of the cloned, amplified fragments was confirmed by DNA sequencing using vector-specific primers. The sequences of the amplified fragments from the two IMAGE clones were found to be identical. The sequence of the encoded protein is shown in Fig. 1. The cloned cDNA was expressed in E. coli BL21-codon plus strain. Cells were grown at 37 °C in LB medium to a density equivalent to A 600 of 0.5–0.7 and induced with 1 mm IPTG for 3 h. However, no recombinant protein could be detected. Induction of protein expression resulted in rapid cessation of cell growth, suggesting that expression of this protein interferes with the host cell metabolism. Lowering the IPTG concentration or growth temperature, reducing the induction time, or using alternative host strains and expression vectors did not improve expression. To overcome this difficulty, the ACPmit gene was re-amplified using two sets of primers, ACPmitT2/B2 (contains the putative mitochondrial targeting sequence) and ACPmitT3/B2 (no targeting sequence) (see Table I). The two amplified fragments were cloned into a modified baculoviral transfer vector pFast Bac 1 (Invitrogen), using appropriate restriction sites that had been engineered into the primers. Coding sequences for a C-terminal His6 tag were engineered into these constructs to facilitate subsequent protein purification by metal ion affinity chromatography. Authenticity of the DNA sequences was confirmed, recombinant baculoviral stocks were generated, and the protein expressed in Sf9 insect cells using the Bac-To-Bac baculoviral expression system (Invitrogen). The recombinant ACPmit expressed with the mitochondrial targeting sequence present was found predominantly in the mitochondrial pellet, both as processed and unprocessed protein. In contrast, the recombinant ACPmit expressed without the targeting sequence was found to be expressed exclusively in the cytosol. For purification of the cytosolic form, insect cells were homogenized at 4 °C in buffer A (50 mm HEPES, pH 8.0, 300 mm NaCl, 10% glycerol) and centrifuged at 4 °C, 144,000 × g for 60 min. The supernatant was filtered (0.45 μm) and loaded onto a HiTrap Chelating HP column (5 ml bed volume) at 20 °C. The column was washed with 50 ml of buffer B (5 mm imidazole in buffer A). The bound proteins were eluted with a 125-ml gradient of 5–125 mm imidazole in buffer A, followed by a 25-ml gradient of 125–250 mm imidazole in buffer A. Fractions containing the enzyme were pooled and the sample buffer was changed to buffer C (50 mm Tris-HCl, pH 7.5, 10% glycerol) at 4 °C, using a Vivaspin concentrator (5,000 M r cutoff). ACPmit was further purified at 20 °C by HiTrap Q HP anion exchange column (1 ml bed volume; Amersham Biosciences) equilibrated with buffer C. The bound proteins were eluted with a 20-ml gradient of 0–250 mm NaCl in buffer C followed by a 5-ml gradient of 250–500 mm NaCl in buffer C. Fractions were analyzed by SDS-PAGE, and fractions containing the apo or holo form were stored separately at –80 °C. Expression of the Peptidyl Carrier Protein (PCP) and Acyl Carrier Protein (ACP-A) from B. brevis and B. subtilis, Respectively—E. coli cells harboring plasmids encoding C-terminally His6-tagged versions of ACP-A (M15 pQE60[acpA]; Ref. 15Mootz H.D. Finking R. Marahiel M.A. J. Biol. Chem. 2001; 276: 37289-37298Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and PCP (M15 pQE70[TycC3PCP]; Ref. 21Weber T. Baumgartner R. Renner C. Marahiel M.A. Holak T.A. Struct. Fold. Des. 2000; 8: 407-418Abstract Full Text Full Text PDF Scopus (162) Google Scholar) were obtained from Dr. Mohamed A. Marahiel at the Institut für Biochemie, Fachbereich Chemie, Philipps-Universität Marburg, Germany. Cells were grown at 37 °C to a density equivalent to A 600 of 0.7, and protein expression was induced with 1 mm IPTG for 3–4 h at either 37 °C (M15) or 30 °C (BL21). Proteins were purified by affinity chromatography by nickel ion affinity chromatography. Assay of Human PPTase Activity—Typical assay systems contained 75–83 mm Tris-HCl, pH 7, 10 mm MgCl2, 66–100 μm [1-14C]acetyl-CoA (or CoASH), 20 μm human apo-ACP, and 0.05–2 μg of human PPTase. Incubations were carried out at 37 °C for 2–5 min. Proteins were precipitated with acid, washed, and assayed for radioactivity. In those experiments where CoASH was used in place of [1-14C]acetyl-CoA, samples were processed for electrospray mass spectrometry. Analysis of PPTase by Gel Filtration Chromatography—To determine the molecular mass of native PPTase, a purified preparation (10 μg) was applied to a BioSep-SEC-S 3000 gel filtration column (300 × 7.8 mm, Phenomenex, Torrance, CA.), equilibrated and eluted at 20 °C with 50 mm Tris-HCl, pH 7.0, 50 mm KCl, 10 mm MgCl2 at a flow rate of 1 ml/min. The column was calibrated with various protein molecular mass standards. Electrospray Mass Spectrometry—Sample was desalted using a C4 ZipTip (Millipore, Bedford, MS), mixed with the matrix (sinapinic acid, 10 mg/ml in 50% acetonitrile plus 0.1% trifluoracetic acid) in a 1:1 ratio (v/v), and 1 μl of the resulting mixture was deposited on the top of pre-crystallized matrix and spotted onto a stainless steel matrix-assisted laser desorption ionization plate. Analysis was performed on a Voyager-DE™ STR matrix-assisted laser desorption ionization time-of-flight biospectrometry workstation (Applied Biosystems, Foster City, CA) in a positive linear mode. Mass scale was externally calibrated utilizing bovine insulin and horse heart myoglobin. Singly charged ions were used for a molecular mass determination. Reported masses represent averages of the results of six acquisitions, and they have been determined within a standard deviation of 1–2 Da. Tissue Specificity of Expression of Human PPTase—A 32P-labeled probe was synthesized by the random priming method using [α-32P]dCTP (3000 Ci/mmol), a PCR-amplified PPTase template cDNA, and a NEBlot kit (New England Biolabs, Beverly, MA). A human multiple tissue Northern blot (Clontech) was probed with the purified radiolabeled probe (∼2 × 106 dpm/blot), using ExpressHyb hybridization solution (Clontech). The amount of poly(A)+ RNA in each lane of the Clontech blot (∼2 μg) has been adjusted to obtain a consistent signal for a housekeeping gene (β-actin) across all lanes. The blot was analyzed using a phosphorimager (Storm 840, Amersham Biosciences). Subcellular Fractionation—Cells were harvested by centrifugation at 600 × g for 5 min, washed with phosphate-buffered saline, and resuspended in 5 volumes of mitochondrial isolation buffer (210 mm mannitol, 70 mm sucrose, 5 mm HEPES-KOH, pH 7.35, 1 mm EDTA) containing a protease inhibitor mixture (EDTA-free protease inhibitor cocktail, Roche Applied Science). The cellular suspension was disrupted, using a Teflon/glass homogenizer, and centrifuged at 800 × g for 10 min at 4 °C to remove cell debris. The supernatant was then centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was collected and further centrifuged at 104,000 × g for 30 min at 4 °C, and the mitochondrial pellet was washed twice with isolation buffer and resuspended in 100 μl of the same buffer. The mitochondria were then ruptured by three cycles of freezing and thawing and the addition of Lubrol (0.1 mg/mg of protein). The 104,000 × g supernatant (cytosol) was collected, and the pellet was resuspended in 200 μl of isolation buffer. Protein concentrations of all three subcellular fractions were determined by the Bradford assay and the fractions stored at –80 °C. The activity of citrate synthase, a mitochondrial matrix enzyme, was assayed (22Alp P.R. Newsholme E.A. Zammit V.A. Biochem. J. 1976; 154: 689-700Crossref PubMed Scopus (290) Google Scholar) in each subcellular fraction to assess the possibility that mitochondria had been damaged during preparation of the homogenate. Cloning and Expression of a Human PPTase—Initial BLAST searches of the GenBank™ sequence data base, using the sequences of various PPTases of the small molecular mass PPTase type, did not reveal a likely candidate for a human PPTase enzyme. However, BLAST searching with a PPTase probe sequence of the high molecular weight type, namely the B. subtilis surfactin synthetase-activating enzyme, Sfp, revealed a candidate human PPTase sequence that was subsequently used to derive full-length cDNAs, by PCR, from human liver and brain cDNA libraries and from a human EST cDNA clone. The putative human PPTase cDNAs obtained from the three sources had identical DNA sequences. When expressed in E. coli, this cDNA generated the expected protein, but it was recovered entirely in inclusion bodies. Expression as a baculoviral vector in Sf9 cells, however, yielded a soluble protein of the expected molecular mass (∼39 kDa, including His6 and TEV protease recognition site) that could be readily purified to homogeneity by a combination of nickel-nitrilotriacetic acid affinity chromatography and anion exchange HPLC (Fig. 2A). Analysis of the purified protein by gel filtration under non-denaturing conditions revealed that the enzyme is a monomer (Fig. 3). The typical yield of purified protein was 5 mg/15 g of cells.Fig. 3Estimation of the molecular mass of native human PPTase by gel filtration. The standards are amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumen (66 kDa), thioesterase I (32 kDa), malonyl/acetyltransferase (35 kDa), and ACPfas (13 kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cloning and Expression of the ACP Domain of the Human Cytosolic FAS—A cDNA encoding the ACP domain of human FAS (ACPfas, residues 2117–2205) was amplified from a human liver cDNA library, cloned into the pQE80 expression plasmid, and its sequence verified. When transfected into E. coli, this cDNA was expressed as a soluble protein (typically 4–8 mg/liter of culture medium) that was readily purified to homogeneity by a combination of nickel ion affinity chromatography and gel filtration (Fig. 2B). Analysis by electrospray mass spectrometry confirmed that, as anticipated, the ACPfas was expressed as the apo form, lacking the phosphopantetheine moiety (Table II). Most of the ACPfas purified as a monomer, but a small portion had a mass corresponding to that of a covalently linked dimer. In the presence of
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