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• W2020552116 abstract "Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content. Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content. Mammalian membranes are enriched with phosphatidylcholine (PtdCho), 2The abbreviations used are: PtdChophosphatidylcholineCCTcytidylyltransferaseCDP-cholinecytidine diphosphocholineCFPcyan fluorescent proteinCHMcycloheximideCKcholine kinaseCPTcholinephosphotransferaseDPPtdChodipalmitoylphosphatidylcholineKGFkeratinocyte growth factorLPCATacyl-CoA:lysophosphatidylcholine acyltransferaseMLEmurine lung epithelialYFPyellow fluorescent proteinβGTUDP-Gal:β-GlcNAcβ1,4-galactosyltransferaseLysoPtdCholysophosphatidylcholineHAhemagglutininCMVcytomegalovirusqRT-PCRquantitative real-time PCR. a zwitterionic phospholipid that serves as a major component of various secretory products, including bile, high density lipoproteins, and pulmonary surfactant. With the exception of hepatic tissues, where PtdCho synthesis may involve sequential N-methylation of phosphatidylethanolamine, PtdCho biosynthesis in mammalian cells occurs primarily via the CDP-choline or de novo pathway. This pathway requires three enzymes: choline kinase (CK) (EC 2.7.1.32), which catalyzes the first committed step; CTP:phosphocholine cytidylyltransferase (CCT) (EC 2.7.7.15), the penultimate enzyme; and cholinephosphotransferase (CPT) (EC 2.7.8.2), which catalyzes the terminal reaction within the CDP-choline pathway generating PtdCho (1.Jackowski S. Fagone P. J. Biol. Chem. 2005; 280: 853-856Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Of these enzymes, CCT is rate-limiting and rate-regulatory. CCT is an amphitrophic enzyme and thus can switch between an inactive soluble or cytoplasmic form and an active, membrane-bound species within the nucleus. There are four isoforms of CCT, and deficiency of a major species, CCTα, is associated with impaired cell growth, apoptosis, and embryonic lethality (1.Jackowski S. Fagone P. J. Biol. Chem. 2005; 280: 853-856Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). CK may also be critical for embryonic development, and genomic deletion of the CK gene results in bone deformities and muscular dystrophy (2.Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 3.Wu G. Aoyama C. Young S.G. Vance D.E. J. Biol. Chem. 2008; 283: 1456-1462Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). CPT (CPT1) catalyzes the transfer of the phosphocholine moiety from CDP-choline to diacylglycerol. A second human choline/ethanolaminephosphotransferase (CEPT1) that displays dual specificity using either CDP-choline or CDP-ethanolamine as substrates with significant primary sequence identity to CPT1 may also regulate PtdCho availability (4.Henneberry A.L. Wistow G. McMaster C.R. J. Biol. Chem. 2000; 275: 29808-29815Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). CPT1 exists in the Golgi, whereas CEPT1 is detected within the endoplasmic reticulum (5.Henneberry A.L. Wright M.M. McMaster C.R. Mol. Biol. Cell. 2002; 13: 3148-3161Crossref PubMed Scopus (163) Google Scholar). The substrate requirements of CPT1 and CEPT1 for diacylglycerol would impact the molecular species profile of the newly synthesized PtdCho. Genetic inactivation of CPT genes results in reduced phospholipid synthesis, and its inhibition by isoprenoids and ceramides triggers apoptosis, underscoring CPT as a key regulator of PtdCho synthesis (6.McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 7.Wright M.M. Henneberry A.L. Lagace T.A. Ridgway N.D. McMaster C.R. J. Biol. Chem. 2001; 276: 25254-25261Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 8.Bladergroen B.A. Bussière M. Klein W. Geelen M.J. Van Golde L.M. Houweling M. Eur. J. Biochem. 1999; 264: 152-160Crossref PubMed Scopus (37) Google Scholar). Unlike CCT, there is limited information on the molecular control of CK, CPT1, and CEPT1. phosphatidylcholine cytidylyltransferase cytidine diphosphocholine cyan fluorescent protein cycloheximide choline kinase cholinephosphotransferase dipalmitoylphosphatidylcholine keratinocyte growth factor acyl-CoA:lysophosphatidylcholine acyltransferase murine lung epithelial yellow fluorescent protein UDP-Gal:β-GlcNAcβ1,4-galactosyltransferase lysophosphatidylcholine hemagglutinin cytomegalovirus quantitative real-time PCR. In addition to the CDP-choline pathway, the generation of PtdCho in tissues involves its remodeling. In the lung, this remodeling mechanism constitutes a major route for generation of dipalmitoylphosphatidylcholine (DPPtdCho), the major component of surfactant that lowers alveolar surface tension (9.den Breejen J.N. Batenburg J.J. van Golde L.M. Biochim. Biophys. Acta. 1989; 1002: 277-282Crossref PubMed Scopus (23) Google Scholar). In this pathway, a phospholipase A2 deacylates PtdCho at the sn-2-position of the glycerol backbone, typically releasing an unsaturated fatty acid. The resultant lysophosphatidylcholine (LysoPtdCho) is then reacylated with a saturated fatty acid (typically 16:0 or 14:0), generating surfactant DPPtdCho. The identity of the deacylating phospholipase A2 is a calcium-independent, acid pH-optimal lysosomal enzyme (aiPLA2) (10.Fisher A.B. Dodia C. Am. J. Physiol. 1997; 272: L238-L243PubMed Google Scholar). The identity of the enzyme that catalyzes the reacylation step remained elusive, but it recently was shown to be an LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) (11.Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11724-11729Crossref PubMed Scopus (149) Google Scholar, 12.Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). A conserved HX4D motif is necessary for enzymatic activity (13.Heath R.J. Rock C.O. J. Bacteriol. 1998; 180: 1425-1430Crossref PubMed Google Scholar), and LPCAT1 expression increases with lung development and in response to glucocorticoids (11.Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11724-11729Crossref PubMed Scopus (149) Google Scholar). LPCAT1 mRNA levels have also been shown to be regulated by keratinocyte growth factor (KGF) in cultured rat type II cells (11.Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11724-11729Crossref PubMed Scopus (149) Google Scholar). The biochemical and molecular regulation of LPCAT1 and its role in PtdCho synthesis in vivo are largely unknown. Lung epithelia must tightly balance levels of surfactant (DPPtdCho) lipid secreted into airways versus nonsurfactant PtdCho destined for membranes. Although the de novo pathway generates both nonsurfactant PtdCho and DPPtdCho, the remodeling pathway would be predicted to synthesize primarily surfactant. Thus, it is possible that cross-talk between these pathways exists to modify pulmonary lipid composition, depending on cellular needs. In other systems, such interdependency exists between lipogenic pathways. For example, rates of PtdCho synthesis are coupled to its degradation (14.Walkey C.J. Kalmar G.B. Cornell R.B. J. Biol. Chem. 1994; 269: 5742-5749Abstract Full Text PDF PubMed Google Scholar, 15.Tijburg L.B. Nishimaki-Mogami T. Vance D.E. Biochim. Biophys. Acta. 1991; 1085: 167-177Crossref PubMed Scopus (17) Google Scholar, 16.Tercé F. Record M. Tronchère H. Ribbes G. Chap H. Biochim. Biophys. Acta. 1991; 1084: 69-77Crossref PubMed Scopus (18) Google Scholar). Overexpression of CCT in COS cells increases PtdCho mass only modestly yet triggers a 3-fold increase in its degradation rate (14.Walkey C.J. Kalmar G.B. Cornell R.B. J. Biol. Chem. 1994; 269: 5742-5749Abstract Full Text PDF PubMed Google Scholar). This coupling between biosynthetic and degradative pathways indicates close regulation of PtdCho metabolism, perhaps as a means to avoid cellular lipotoxicity. Further, overexpression of PEMT2 (phosphatidylethanolamine N-methyltransferase-2) in hepatoma cells results in feedback inhibition of CCT by reducing its transcriptional rate without altering PtdCho content (17.Cui Z. Houweling M. Vance D.E. Biochem. J. 1995; 312: 939-945Crossref PubMed Scopus (43) Google Scholar). These latter data suggest interdependency between the N-methylation and CDP-choline pathways for PtdCho biosynthesis. The relationship between the remodeling and de novo pathways for PtdCho synthesis has not been investigated. Herein, we hypothesized that the remodeling pathway regulates the CDP-choline pathway to maintain PtdCho balance. Overexpression of the remodeling enzyme, LPCAT1, in lung epithelia significantly decreased de novo PtdCho synthesis without altering cellular PtdCho levels. LPCAT1 expression increased degradation of the final enzyme within the CDP-choline pathway, CPT1, a multiubiquitinated enzyme, through its lysosomal elimination. CPT mutants harboring arginine substitutions at multiple putative ubiquitination acceptor sites conferred proteolytic resistance to inhibitory effects of LPCAT1 expression in cells. The data provide new insight into the molecular processing of a key regulatory enzyme involved in PtdCho biosynthesis and are the first indicating interdependence between remodeling and de novo pathways to preserve lipid homeostasis. The sources of murine lung epithelial (MLE) cells, culture medium, immunoblotting materials, and radiochemicals were described previously (18.Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Rat primary alveolar type II epithelia were isolated as described (19.Salome R.G. McCoy D.M. Ryan A.J. Mallampalli R.K. J. Appl. Physiol. 2000; 88: 10-16Crossref PubMed Scopus (24) Google Scholar). Mouse monoclonal ubiquitin antibody was purchased from Cell Signaling (Danvers, MA). The pAmCyan1-C1 and pZsYellow-C1 vector was purchased from Clontech. LysoTracker Red; mouse monoclonal V5 antibody; the To-Pro-3 nuclear staining kit; the PCRTOPO4.1 cloning kit; pcDNA-DEST40, pcDNA3.1/nV5-DEST, and pLenti6/V5-Dest cloning vectors; Escherichia coli One Shot competent cells; the pENTR Directional TOPO cloning kits; LR Clonase II recombinase; the Superscript III RT kit; and the Gateway mammalian expression system were purchased from Invitrogen. Ni2+ resin, HIS-select nickel affinity gel, Tri Reagent®, human KGF, and β-actin primary mouse monoclonal antibody were obtained from Sigma. LPCAT antibody was generated by Covance (Princeton, NJ). Amicon Ultra-4 centrifugal filter devices were from Millipore (Billerica, MA). The QuikChangeTM site-directed mutagenesis kit, XL-gold cells, and pCMV-Tag1 vector were from Stratagene (La Jolla, CA). A ubiquitin plasmid was constructed as described (20.Chen B.B. Mallampalli R.K. Mol. Cell. Biol. 2009; 29: 3062-3075Crossref PubMed Scopus (46) Google Scholar). The gel extraction kit and QIAprep Spin Miniprep kits were from Qiagen (Valencia, CA). NucleoBond Xtra Maxi prep kits were obtained from Macherey-Nagel (Bethlehem, PA). Cyclohexamide (CHM) and UbiQapture-Q matrix were from Biomol (Plymouth Meeting, PA). HA-tagged ubiquitin was a gift from Dr. Peter Snyder (University of Iowa). β-1,3-Galactosyltransferase 2 goat polyclonal primary antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Power CYBR Green PCR master mix was from Applied Biosystems (Carlsbad, CA). All restriction enzymes and ligases were purchased from New England Biolabs (Ipswich, MA). The TNT coupled reticulocyte lysate system and RQ1 DNase kit were purchased from Promega (Madison, WI). All DNA sequencing was performed by the University of Iowa DNA Core Facility. Cloning primers were purchased from IDT (Coralville, IA). The Zeiss LSM 510 confocal microscope is part of the University of Iowa Central Microscopy Research Facility. RNA was purified from primary murine lung cells (21.Zhou J. Wu Y. Henderson F. McCoy D.M. Salome R.G. McGowan S.E. Mallampalli R.K. Gene Ther. 2006; 13: 974-985Crossref PubMed Scopus (15) Google Scholar). Total cellular RNA was isolated using Tri Reagent, and cDNA was obtained by reverse transcription followed by DNase I digestion, amplification, and detection by a Chromo 4 real-time PCR detector (22.Chen B.B. Mallampalli R.K. J. Biol. Chem. 2007; 282: 33494-33506Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Levels of transcripts were measured relative to GAPDH or 18 S mRNA. The coding sequence available for LPCAT1, CPT1, and CEPT1 on the NCBI Web site (NM_145376, NM_144807, NM_133869) were used to construct primers for cloning of genes from cDNA from mRNA via reverse transcriptase PCR from murine liver and kidney tissues. Amplified fragments were subcloned into PCRTOPO4.1, sequenced, and were found to be identical to the deposited NCBI LPCAT1 sequence. The resulting PCRTOPO4.1 vector served as a source for cloning into pacAd5 CMV internal ribosome entry site enhanced green fluorescent protein pA (University of Iowa DNA Core) using ClaI and BamHI restriction sites. For CPT1 and CEPT1, the Invitrogen Gateway system was used. Primers constructed containing CACC overhangs upstream of the 5′ ATG and antisense sequence with (V5-CEPT1) or without (CPT-V5his) a stop codon were used for amplification using a blunt end polymerase and pENTR/D-TOPO per the manufacturer's instructions. LR Clonase II recombinase was used for cloning of CPT1 and CEPT1 sequences into pcDNA-DEST40 or pcDNA3.1/nV5-DEST gateway vectors, respectively (CPT1-V5his, V5-CEPT1). CPT1K4R and CPT1K6R were constructed by performing site-directed mutagenesis on CPT1-V5his at Lys254, Lys282, Lys283, and Lys292 (CPT1K4R) and additionally Lys307 and Lys311 for CPT1K6R using the QuikChange II XL site-directed mutagenesis (Stratagene) kit. CFP-CPT1 was generated, similar to CFP-CCT (22.Chen B.B. Mallampalli R.K. J. Biol. Chem. 2007; 282: 33494-33506Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The CPT1 construct CPT1K6R was digested with BglII and SalI, purified, and ligated into pAmCyan-C1 as described, generating CFP-CPT1K6R (20.Chen B.B. Mallampalli R.K. Mol. Cell. Biol. 2009; 29: 3062-3075Crossref PubMed Scopus (46) Google Scholar). FLAG-CPT1 was constructed by amplifying CPT1 and ligation into pCMV-Tag1. A βGT-YFP (carboxyl-terminal YFP tag) plasmid was constructed by amplifying or digesting three separate fragments for ligation. First, a human cDNA was used as a template to amplify the first 249 base pairs of the UDP-Gal:βGlcNAcβ1,4-galactosyltransferase gene (B4GALT1) (GenBankTM number BC045773) with flanking ClaI/EcoRV restriction sites. Fragment 2 was an EcoRV/EcoRI YFP fragment amplified from pZsYellow-C1. Finally a pacAd5 CMV internal ribosome entry site enhanced green fluorescent protein pA vector, and the two amplified fragments were digested with appropriate restriction enzymes and gel-extracted. The B4GALT1 and YFP fragments were ligated into the pacAd5 CMV internal ribosome entry site enhanced green fluorescent protein pA to generate βGT-YFP. Tandem Ubi-CFP-CPT fusion constructs were generated by cloning the ubiquitin coding sequence at the carboxyl terminus of CFP-CPT1 using SalI and ApaI sites to generate CFP-CPT1×Ub, CFP-CPT3×Ub, and CFP-CPT4×Ub. Alveolar macrophages, primary type II cells, and fibroblasts were isolated as described previously (23.McCoy D.M. Fisher K. Robichaud J. Ryan A.J. Mallampalli R.K. Am. J. Respir. Cell Mol. Biol. 2006; 35: 394-402Crossref PubMed Scopus (13) Google Scholar). MLE cells were maintained in Hite's medium (Dulbecco's modified Eagle's medium/F-12 medium) with 2% fetal bovine serum at 37 °C in 5% CO2. After reaching 80% confluence, the cells were harvested using 0.25% trypsin and 0.1% EDTA, resuspended in medium, and plated onto appropriate culture dishes containing a 3 μl/1 μg DNA ratio of FuGENE6 lipofection reagent and appropriate expression vector. After incubation overnight, the medium was replaced with Hite's medium with 2% fetal bovine serum for 8 h before cell harvesting. In some studies, an Amaxa electroporation device with program T-013 and solution L was used for plasmid transfection of cells. Cells were maintained as described above, except after trypsinization, cells were resuspended in a small volume of solution L per the manufacturer's directions. Plasmids were transfected into cells, or cells were left untransfected. Medium was aspirated 24 h post-transfection, and cells were treated an additional 24 h with 0% fetal bovine serum Dulbecco's modified Eagle's medium/F-12 medium supplemented with or without NH4Cl (25 mm). Cell lysates were harvested by brief sonication in 150 mm NaCl, 50 mm Tris-HCl, 1 mm EDTA (no EDTA if Ni2+ purification was performed), 2 mm dithiothreitol, 0.025% sodium azide, and 1 mm phenylmethylsulfonyl fluoride (pH 7.4), at 4 °C. Alternatively, cells were switched to Dulbecco's modified Eagle's medium/F-12 medium without fetal bovine serum and treated for 6 h with CHM (18 μg/ml) with either NH4Cl (25 mm) or lactacystin (25 μm). Cells were collected in Buffer A plus 0.5% Triton X-100 plus 0.5% Nonidet P-40 and sonicated for further analysis. MLE cells were also exposed to human KGF (20 ng/ml) for 24 h prior to harvest. Rat type II cells were transduced for 48 h with lentivirus, constructed at the University of Iowa gene transfer vector core, containing LPCAT1 in a pLenti6/V5-Dest vector. Cells were resuspended in Buffer A, and samples were first centrifuged at 16,000 × g for 10 min at 4 °C. The resulting supernatant was centrifuged at 100,000 × g for 60 min at 4 °C. The resulting microsomal pellet was resuspended in Buffer A using a 25-gauge needle. Lipids were extracted from equal amounts of membrane protein, and levels of the individual phospholipids were quantitated with a phosphorus assay (24.Mallampalli R.K. Walter M.E. Peterson M.W. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1994; 10: 48-57Crossref PubMed Scopus (16) Google Scholar). DPPtdCho was assayed as before (25.Agassandian M. Miakotina O.L. Andrews M. Mathur S.N. Mallampalli R.K. Biochem. J. 2007; 403: 409-420Crossref PubMed Scopus (29) Google Scholar). For PtdCho de novo synthesis, cells were pulsed with 1 μCi of [methyl-3H]choline chloride during the final 2 h of incubation with choline-depleted medium. For remodeling activity, cells were labeled with 1.75 nm [14C]LysoPtdCho (55 mCi/mmol) for 3 h. Total cellular lipids were extracted, resolved using TLC, and processed for scintillation counting (18.Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Cells were harvested in lysis buffer (250 mm sucrose, 10 mm Tris-HCl, pH 7.4), and equal amounts of microsomal cellular protein were used in the assay. 1 nmol of 1-palmitoyl-sn-glycero-3-phosphocholine (50.44 mm solution in 1:1 chloroform/methanol)/μl of 5× assay buffer was added and sonicated with 5× assay buffer (final concentration 65 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 12.5 mm fatty acid-free bovine serum albumin, 2 mm EDTA). 35 μl of H2O, and cellular protein was added to 10 μl of sonicated assay buffer and 5 μl of [1-14C]acyl-CoA (0.1 μCi, 1.8 nmol) for a total assay volume of 50 μl. Upon the addition of [1-14C]acyl-CoA, samples were incubated at 30 °C for 10 min, after which the reaction was terminated by the addition of chloroform/methanol/H2O (1:2:0.70, v/v/v). Total cellular lipids from reaction mixtures were extracted by the method of Bligh and Dyer (26.Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42411) Google Scholar) and spotted onto LK5D plates, and PtdCho was resolved using TLC and detected using a plate reader (18.Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 27.Mallampalli R.K. Salome R.G. Spector A.A. Am. J. Physiol. 1994; 267: L641-L648PubMed Google Scholar). The activity of CK was assayed as described (18.Mallampalli R.K. Ryan A.J. Salome R.G. Jackowski S. J. Biol. Chem. 2000; 275: 9699-9708Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CCT activity was determined by using a charcoal extraction method (28.Mallampalli R.K. Mathur S.N. Warnock L.J. Salome R.G. Hunninghake G.W. Field F.J. Biochem. J. 1996; 318: 333-341Crossref PubMed Scopus (29) Google Scholar). CPT activity was assayed as described (29.Miller J.C. Weinhold P.A. J. Biol. Chem. 1981; 256: 12662-12665Abstract Full Text PDF PubMed Google Scholar). Each reaction mixture contained 50 mm Tris·HCl buffer (pH 8.2), 0.1 mg/ml Tween 20, 1 mm 1,2- dioleoylglycerol, 0.8 mm phosphatidylglycerol, 0.5 mm [14C]CDP-choline (specific activity 1,110 dpm/nmol), 5 mm dithiothreitol, 5 mm EDTA, 10 mm MgCl2, and 30–40 μg of protein. The lipid substrate was prepared by combining appropriate amounts of 1,2-dioleoylglycerol (1 mm) and phosphatidylglycerol (0.8 mm) in a test tube, drying under nitrogen gas, and brief sonication before the addition to the assay mixture to achieve the final desired concentration. The reaction proceeded for 1 h at 37 °C and terminated with 4 ml of methanol/chloroform/water (2:1:7, v/v/v). The remainder of the assay was performed exactly as described (29.Miller J.C. Weinhold P.A. J. Biol. Chem. 1981; 256: 12662-12665Abstract Full Text PDF PubMed Google Scholar). A Bioscan AR-2000 plate reader was used for detection of radiolabeled PtdCho on LK5D TLC plates with data quantified using WinScan software. PtdCho bands on the LK5D silica plates were also scraped and quantified by liquid scintillation counting. Immunoblotting was performed as described (22.Chen B.B. Mallampalli R.K. J. Biol. Chem. 2007; 282: 33494-33506Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 30.Agassandian M. Zhou J. Tephly L.A. Ryan A.J. Carter A.B. Mallampalli R.K. J. Biol. Chem. 2005; 280: 21577-21587Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The dilution factors for the LPCAT1, V5, FLAG, and β-actin antibodies were 1:2000, 1:2000, 1:5000, and 1:10,000, respectively. Cell lysates were also incubated with 3 μl of HA antibody (Sigma) at 4 °C overnight, incubated with 20 μl of Pierce protein A/G matrix for 2 h, and eluted with protein sample buffer/lysis buffer prior to immunoblot analysis (22.Chen B.B. Mallampalli R.K. J. Biol. Chem. 2007; 282: 33494-33506Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 30.Agassandian M. Zhou J. Tephly L.A. Ryan A.J. Carter A.B. Mallampalli R.K. J. Biol. Chem. 2005; 280: 21577-21587Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). MLE cells were plated at 30% confluence on 35-mm MetTek glass bottom culture dishes and transfected with individual CFP plasmids. Cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 min and then exposed to 15% bovine serum albumin, 1:200 β1,3-galactosyltransferase 2 primary goat antibody, and 1:200 fluorescein isothiocyanate-conjugated AffiniPure Donkey anti-goat IgG (H + L) (Jackson Immunoresearch) to visualize the trans-Golgi; cells were also incubated with an Alexa568-labeled goat anti-rabbit secondary antibody. Nuclei were visualized using To-Pro-3 (1:2000 dilution). Immunofluorescent cell imaging was performed on a Zeiss LSM 510 confocal microscope using the 458-, 568-, or 615-nm wavelength. All experiments were done with a Zeiss ×63 or ×100 oil differential interference contrast objective lens. The 458-nm wavelength was used to excite the CFP-CPT1 fusion proteins, with fluorescence emission collected through a 475-nm filter. A 488-nm wavelength was used to excite fluorescein dye, with fluorescence emission collected through a 505–530-nm filter. A 488-nm wavelength was used to excite βGT-YFP, with fluorescence emission collected through a 530–600-nm filter. A 613-nm wavelength was used to excite To-Pro-3 dye, with fluorescence emission collected through a 633-nm filter. Scanning was bidirectional at the highest possible rate measurement using a digital 1× zoom. Cell lysates were incubated with 40 μl of agarose beads complexed to ubiquitin-binding domain peptide overnight at 4 °C. An aliquot of prebound lysate was also resolved by 10% SDS-PAGE to determine loading onto the resin. The matrix resin was washed with 5 × 1 volume of 9:1 phosphate-buffered saline to lysis buffer as described in the Biomol protocol, and protein sample buffer was used to elute bound protein. Cell lysates were incubated with 40 ml of His-select nickel affinity gel resin overnight at 4 °C. The matrix resin was washed sequentially with a 3 × 3 bead volume of Buffer A without EDTA, 0.05% Triton X-100, and 0.05% Nonidet P-40; a 2 × 3 bead volume of Buffer A without EDTA, 0.005% Triton X-100, 0.005% Nonidet P-40, 500 mm NaCl, and 6 mm imidazole; and finally 1 volume of Buffer A without EDTA, 0.005% Triton X-100, 0.005% Nonidet P-40, 500 mm NaCl, and 200 mm imidazole. After 30 min, protein sample buffer was added to the resin/buffer mixture, samples were incubated at 25 °C overnight, and proteins were visualized by immunoblot analysis. Statistical analysis was performed by two-way analysis of variance or Student's t test. Data are presented as mean ± S.E. Lung epithelial type II cells synthesize and secrete surfactant composed of the major surface-active species, DPPtdCho. At least three acytransferases might participate in remodeling of PtdCho within lung epithelia generating DPPtdCho. These proteins include LPCAT1 and two variants harboring similar catalytic domains, termed LPCAT2 (31.Shindou H. Hishikawa D. Nakanishi H. Harayama T. Ishii S. Taguchi R. Shimizu T. J. Biol. Chem. 2007; 282: 6532-6539Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and LPCAT3 (32.Ye G.M. Chen C. Huang S. Han D.D. Guo J.H. Wan B. Yu L. DNA Seq. 2005; 16: 386-390Crossref PubMed Scopus (33) Google Scholar) (AGPAT7 and LPEAT2); these enzymes exhibit similar substrate requirements with regard to saturated (16:0) fatty acyl-CoA donors (33.Soupene E. Fyrst H. Kuypers F.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 88-93Crossref PubMed Scopus (80) Google Scholar). To evaluate which species may represent the primary acyltransferase involved in surfactant remodeling, we isolated mouse epithelial type II cells, alveolar macrophages, and lung fibroblasts. qRT-PCR shows that LPCAT1 mRNA is highly expressed in surfactant-producing mouse primary type II cells, with markedly reduced expression in macrophages or fibroblasts. Lung epithelia also expressed mRNAs encoding LPCAT2 and LPCAT3, albeit at lower levels (Fig. 1, B and C). Of the three homologues, LPCAT2 expression was highly expressed within macrophages, and LPCAT3 was most predominant within fibroblasts (Fig. 1, B and C). Thus, LPCAT1 appears to be a major regulator of surfactant remodeling, but the data do not exclude redundancy with other related acyltransferases. Because LPCAT1 was highly expressed in surfactant-producing lung cells, this enzyme was cloned and expressed in murine lung epithelia to test its regulatory activity on PtdCho metabolism. Using nucleofection, we were able to achieve high level (>90%) transfection efficiency in mammalian epithelia within a strong CMV-driven mammalian expression vector (Adv-CMV-LPCAT1). Following expression of this plasmid, we detected a ∼10-fold increase in LPCAT1 mRNA (Fig. 2A), a 28-fold increase in immunoreactive LPCAT1 (Fig. 2B, inset), a ∼7-fold increase in LPCAT activity (Fig. 2B), and a nearly 3-fold increase in [3H]LysoPtdCho incorporation into DPPtdCho (Fig. 2C). Importantly, despite up-regulating LPCAT1 expression and remodeling activity," @default.
• W2020552116 created "2016-06-24" @default.
• W2020552116 creator A5030631527 @default.
• W2020552116 creator A5060804342 @default.
• W2020552116 date "2010-02-01" @default.
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