JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M600225200 on May 16, 2006

J. Biol. Chem., Vol. 281, Issue 29, 20140-20147, July 21, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/29/20140    most recent
M600225200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakanishi, H.
Right arrow Articles by Shimizu, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakanishi, H.
Right arrow Articles by Shimizu, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cloning and Characterization of Mouse Lung-type Acyl-CoA:Lysophosphatidylcholine Acyltransferase 1 (LPCAT1)

EXPRESSION IN ALVEOLAR TYPE II CELLS AND POSSIBLE INVOLVEMENT IN SURFACTANT PRODUCTION*Formula

Hiroki Nakanishi{ddagger}§, Hideo Shindou§1, Daisuke Hishikawa§, Takeshi Harayama§, Rie Ogasawara, Akira Suwabe, Ryo Taguchi{ddagger}||, and Takao Shimizu§2

From the Departments of {ddagger}Metabolome and §Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the Department of Laboratory Medicine, School of Medicine, Iwate Medical University, 19-1 Uchimaru, Morioka, Iwate 020-8505, and ||CREST of the Japan Science and Technology Agency, Kawaguchi, Saitama, 332-8613, Japan

Received for publication, January 10, 2006 , and in revised form, May 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, PC), is an important constituent of biological membranes. It is also the major component of serum lipoproteins and pulmonary surfactant. In the remodeling pathway of PC biosynthesis, 1-acyl-sn-glycero-3-phosphocholine (LPC) is converted to PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23 [EC] ). Whereas LPCAT activity has been detected in several tissues, the structure and detailed biochemical information on the enzyme have not yet been reported. Here, we present the cloning and characterization of a cDNA for mouse lung-type LPCAT (LPCAT1). The cDNA encodes an enzyme of 60 kDa, with three putative transmembrane domains. When expressed in Chinese hamster ovary cells, mouse LPCAT1 exhibited Ca2+-independent activity with a pH optimum between 7.4 and 10. LPCAT1 demonstrated a clear preference for saturated fatty acyl-CoAs, and 1-myristoyl- or 1-palmitoyl-LPC as acyl donors and acceptors, respectively. Furthermore, the enzyme was predominantly expressed in the lung, in particular in alveolar type II cells. Thus, the enzyme might synthesize phosphatidylcholine in pulmonary surfactant and play a pivotal role in respiratory physiology.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylcholine (PC)3 is a major component of cellular membranes, and an important constituent of serum lipoproteins and pulmonary surfactant. PC is formed from diacylglycerol by a de novo pathway that was originally described by Kennedy in 1961 (1). PC can also be generated by LPC remodeling. The rapid turnover of the sn-2 acyl moiety of glycerophospholipids was described by Lands (Lands' cycle) (2–4), and is attributed to activation of phospholipase A2s and lysophospholipid acyltransferases. Although these metabolic processes are carried out in a variety of different tissues, the enzyme molecule(s) involved in LPC remodeling have not been identified. We previously cloned 1-acyl-sn-glycerol-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT), and found that the enzyme used LPA as an acceptor (5). Whereas the recombinant enzyme utilized various fatty acyl-CoAs as acyl donors, no other lysophospholipid served as an acceptor. Several lysophospholipid acyltransferases have been cloned in the last decade (6–24), and putative LPAATs ({alpha}, beta, {gamma}, {delta}, {epsilon}, and {zeta}) and tafazzins have been identified (5, 12–16, 18, 22–26). However, LPCAT, the most important enzyme in membrane biogenesis and surfactant production, has not yet been isolated. Using previously proposed acyltransferase motifs (27), and an extensive genomic data base search as well as 5'- and 3'-rapid amplification of cDNA ends, we identified a candidate Lpcat gene. The enzyme was highly expressed in the lung, in particular in alveolar type II cells, and it possessed a catalytic preference for the production of di-saturated PC. Thus, we propose that this enzyme plays a critical role in the production of surfactant lipids. To our knowledge, this is the first documentation of a cDNA for LPCAT, a critically important enzyme in the biogenesis of membrane phospholipids and surfactant lipids. We termed the enzyme LPCAT1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
[1-14C]Palmitoyl-CoA (2.22 GBq/mmol), [1-14C]palmitoyl-LPC (2.07 GBq/mmol), and horseradish peroxidase-linked anti-mouse IgG were from Amersham Biosciences. Four amidinophenylmethanesulfonyl fluoride, M2 and M5 anti-FLAG mouse monoclonal antibody, Dulbecco's modified Eagle's medium, and linoleoyl-CoA (C18:2) were purchased from Sigma. A proteinase inhibitor mixture, Complete, was from Roche. TLC silica gel plates (type 5721) were purchased from Merck (Darmstadt, Germany). Alexa Fluor 546 goat anti-mouse IgG, LysoTracker Red DND-99, and MitoTracker Red CMXRos were purchased from Molecular Probes, Inc. (Eugene, OR). An anti-calnexin antibody was from BD Biosciences (Tokyo, Japan). Various lysophospholipids and acyl-CoAs were obtained from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL).

Cloning of Acyl-CoA:LPC Acyltransferase 1 (LPCAT1)
The mouse lysophospholipid acyltransferase gene was identified based upon sequence similarity to the C terminus of the Lpaat{zeta} gene through a comprehensive BLAST search. A 1.6-kb cDNA clone encoding the full-length mouse Lpcat1 (LPCAT1, DDBJ accession number AB244717 [GenBank] ) was cloned by PCR amplification using the forward primer 5'-CTAGCTAGCCACCATGGATTACAAGGATGACGATGACAAGAGGCTGCGGGGCCGCGGGCCGCGG-3', the reverse primer 5'-CCGCTCGAGCTAGTCCGCTTTCTTACAAGAATTC-3', and a mouse spleen cDNA library as a template. The FLAG epitope (DYKD-DDDK) was attached to the N terminus of mLpcat1 using PCR, and a forward primer that encoded the FLAG sequence inframe with the start codon of the mLpcat1 coding region. Amplification was carried out for 5 cycles of 96 °C for 15 s, 40 °C for 20 s, and 68 °C for 1.75 min, followed by 30 cycles of 96 °C for 15 s, 60 °C for 20 s, and 68 °C for 1.75 min. Amplified PCR products were cloned into the pCXN2.1 vector (28) and sequenced. Similarly, human and rat LPCAT1 cDNAs (DDBJ accession numbers AB244719 [GenBank] and AB244983 [GenBank] , respectively) were amplified by PCR and inserted into the pCXN2.1 vector.

Quantitative Real-time RT-PCR
Mouse total RNA was prepared from various tissues using Absolutely RNA Miniprep Kit (Stratagene, La Jolla CA), and first strand cDNAs were synthesized using Superscript II (Invitrogen, Tokyo, Japan). PCR were carried out in microcapillary tubes, in 20-µl reaction volumes consisting of 2 µl of cDNA solution, 1x FastStart DNA Master SYBR Green I (Roche Applied Science), and 0.5 µM each of the forward and reverse primer. A 163-bp fragment of mLpcat1 or rat Lpcat1 was generated using the following primers: forward primer, 5'-GTGCACGAGCTGCGACT-3'; reverse primer, 5'-GCTGCTCTGGCTCCTTATCA-3'. A 227-bp fragment of rat surfactant protein-C was generated using the following primers: forward primer, 5'-CTCCACTGGCATCGTTCT-3'; reverse primer, 5'-CTCGCCCAGAAGAATCAG-3'.

Expression of FLAG-mLPCAT1 in CHO-K1 Cells
FLAG-tagged mLpcat1 was constructed by ligating the FLAG-tagged cDNA (as described above) into the NheI-XhoI site of pCXN2.1. DNA transfection of CHO-K1 cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and cells were harvested 48 h post-transfection.

Confocal Microscopy
CHO-K1 cells were seeded onto 6-cm dishes at a density of 8 x 105 cells/dish 1 day before transfection. Six µg each of pCXN2.1 vector or FLAG-mLpcat1-pCXN2.1 were transfected using Lipofectamine 2000. 48 h post-transfection, pCXN2.1 vector- or FLAG-mLPCAT1-pCXN2.1-transfected cells were incubated with 2.5 µg/ml DiOC6(3) (Invitrogen) and 10 µg/ml M5 anti-FLAG mouse monoclonal antibody (Sigma) in 1/4x Permeabilization Buffer (Beckman). After washing, cells were incubated with 10 µg/ml Alexa Fluor 546 goat anti-mouse IgG (Eugene, OR) for 30 min. pEGFP-C1 vector- or pEGFP-mLP-CAT1-transfected cells were stained with 100 nM MitoTracker Red CMXRos or 50 nM LysoTracker Red DND-99 in Hanks' balanced salt solution containing 10 mM HEPES (pH 7.4) and 0.1% bovine serum albumin at 37 °C for 30 min. Confocal microscopy was performed with an LSM510 Laser Scanning Microscope (Carl Zeiss, Germany) equipped with a 63x water-immersion objective lens (NA = 1.2). FLAG-mLPCAT1, MitoTracker Red, and LysoTracker Red were monitored by excitation at 543 nm with a He/Ne laser, and by emission with a 585-nm long path filter. For the detection of DiOC6(3) and GFP fluorescence, the excitation was at 488 nm with an argon laser, and emissions were taken with a 505–550-nm band pass filter.

Western Blot Analysis
One µg of total cellular protein was resolved by 10% SDS-PAGE and transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% skim milk, incubated with M2 anti-FLAG mouse monoclonal antibody, washed, and then incubated with horseradish peroxidase-linked anti-mouse IgG (Amersham Biosciences). After washing, the membranes were exposed to ECL reagents (Amersham Biosciences) and x-ray film (Amersham Biosciences) to visualize immunoreactive proteins. 7 µg of total cellular protein was analyzed by Western blot using an anticalnexin antibody as an endoplasmic reticulum (ER) marker.

Assay of Lysophospholipid Acyltransferase
For the preparation of cell extracts, cells in 10-cm dishes were scraped into 1 ml of ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 300 mM sucrose, and a proteinase inhibitor mixture, Complete, then sonicated three times on ice for 30 s. After centrifugations for 10 min at 800 and 9,000 x g, the supernatant was collected and centrifuged at 100,000 x g for 1 h. The resulting pellet was resuspended in buffer containing 20 mM Tris-HCl (pH 7.4), 300 mM sucrose, and 1 mM EDTA. Protein concentration was measured by the method of Bradford (29), using a protein assay solution (Bio-Rad) and bovine serum albumin (fraction V, fatty acid-free, Sigma) as a standard. The acyltransferase activity was measured in two ways: (i) conversion of [1-14C]palmitoyl-LPC (293 MBq/mmol) to PC in the presence of acyl-CoA and (ii) the transfer of [1-14C]palmitoyl-CoA (293 MBq/mmol) to lysophospholipids to form phospholipids. Reaction mixtures contained 100 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mg/ml PC, the indicated concentrations of acyl-CoA and lysophospholipid, and enzyme in a total volume of 0.1 ml. After incubation at 30 °C for 5 min, reactions were stopped by the addition of 0.3 ml of chloroform:methanol (1:2, v/v). Total lipid was extracted by the Bligh-Dyer method (30), and subsequently analyzed by TLC in chloroform:methanol:acetic acid: water (50:25:8:4, v/v). Bands at positions corresponding to the expected product were visualized by I2 vapor, cut off the plate, placed in Microscinti-O (PerkinElmer Life Sciences), and analyzed in a liquid scintillation counter LS6500 (Beckman).


Figure 1
View larger version (88K):
[in this window]
[in a new window]
  		FIGURE 1.
Amino acid sequence alignment of mLPCAT1 with human and rat LPCAT1. The predicted conserved lysophospholipid acyltransferase motifs (motifs 1, 2, and 3) are boxed. The putative transmembrane domains are underlined. Amino acids conserved among the three species are marked with asterisks (*). A sequence motif (KKXX) predicted to be involved in ER localization is present at the extreme C terminus. The transmembrane motifs were determined using HMMTOP (50).

 
Isolation of Rat Alveolar Type II Cells
Cells were isolated by the method of Dobbs and Mason (31). In brief, 6–8-week-old Sprague-Dawley (200–250 g body weight) rats were injected intraperitoneally with heparin and pentobarbital. The heart-lung preparation was perfused with buffer A consisting of 140 mM NaCl, 5 mM KCl, 2.5 mM sodium potassium phosphate, 10 mM HEPES (pH 7.4), 1 mg/ml glucose, and 10 mg/ml gentamycin. The lungs were removed en block from the thoracic cavity and washed five times with buffer A. Lavage fluids were collected and centrifuged to obtain alveolar macrophages, which were used as a negative control in these experiments. After washing, a fluorocarbon (Sumitomo 3M, Tokyo, Japan)-albumin emulsion was injected intratracheally, and the lung was incubated at 37 °C for 30 min. This step facilitated the separation of alveolar macrophages from alveolar type II cells. After extensive washing with buffer containing 2 mM EGTA, the lung was injected intratracheally with about 40 ml of elastase solution (porcine pancreas elastase, 4.3 units/ml, Worthington, Lakewood, NJ). The lung was minced into 1-mm3 pieces, shaken vigorously during incubation at 37 °C for 5 min, and cells released from the lung were recovered through nylon gauzes. Type II alveolar epithelial cells were isolated from the upper layer of a metrizamide density gradient, washed with buffer A containing 2 mM CaCl2 and 1.3 mM MgSO4, and incubated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The fraction contained >90% alveolar type II cells. These cells were quickly frozen, and subsequently used for RT-PCR or enzymatic analyses.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2.
Tissue distribution of mLpcat1 mRNA. The expression levels of mLPCAT1 and beta-actin mRNA in 19 mouse tissues were analyzed by quantitative RT-PCR. The levels of mRNA were normalized to those of beta-actin mRNA in each tissue. The highest level of mLpcat1 expression was observed in the lung. A similar result was obtained in an independent experiment.

 
Microscopic Analyses
Transmission Electron Microscopy—Alveolar type II cells freshly isolated from rat lungs were seeded on aclar film (Nisshin EM, Tokyo, Japan) and incubated overnight. The cells were then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After a brief wash with the same buffer, postfixation with 1% OsO–4 in 0.1 M phosphate buffer was carried out for 2 h, followed by gradual dehydration with ethanol. The cells were embedded in Epon. Thin serial sections (90-nm thickness) were stained with uranyl acetate and lead citrate. The samples were examined at 100 kV with a HITACHI H-7100s (HITACHI, Tokyo, Japan).

Differential Interference Contrast Microscopy—2 x 106 rat alveolar type II cells were seeded on a 35-mm glass-bottomed dish (IWAKI) and incubated overnight. The cells were rinsed with Dulbecco's modified Eagle's medium and mounted into a CO2 incubator placed on the stage of an inverted microscope (Carl Zeiss LSM 510 system) 30 min before the observation.

Animals
All animal studies were conducted in accordance with the guidelines for animal research at The University of Tokyo and were approved by The University of Tokyo Ethics Committee for Animal Experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Cloning of LPCAT1—The mouse Lpcat1 gene was identified based upon sequence homology with previously reported lysophospholipid acyltransferases, including a putative mouse Lpaat{zeta} gene. A 1.6-kb cDNA clone encoding the full-length mouse LPCAT1 enzyme was obtained by PCR amplification. The putative open reading frame of mLPCAT1 encoded a 534-amino acid protein of 59.8 kDa, containing three transmembrane domains and several conserved motifs found in members of the lysophospholipid acyltransferase family (5, 12–16, 20, 21, 26, 27). The presence of a C-terminal sequence motif (KKXX) indicated that the enzyme localizes to the ER (32). mLPCAT1 exhibited 88.2 and 98.1% amino acid sequence identity to human and rat LPCAT1, respectively (Fig. 1). The protein contained a putative EF-hand-like motif (from amino acids 380 to 480) (33), but we found no obvious Ca2+ requirement for catalytic activity (see below).


Figure 3
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3.
Subcellular localization of FLAG- or EGFP-mLPCAT1 in CHO-K1 cells. 48 h post-transfection, CHO-K1 cells were subjected to immunocytochemical (A) and Western blot (B) analysis. A, ER-Golgi, mitochondria, and lysosome were visualized using DiOC6(3) (green), MitoTracker Red (red), and LysoTracker Red (red), respectively. FLAG-mLPCAT1 was visualized using M5 anti-FLAG peptide antibody as shown in red, and EGFP-mLPCAT1 was observed as green color. The scale bars correspond to 20 µm. B, the 9,000 x g pellet, 100,000 x g pellet, and 100,000 x g supernatant fractions from transfected CHO-K1 cells were subjected to Western blot analysis using M2 anti-FLAG antibody or anti-calnexin antibody as an ER marker. FLAG-mLPCAT1 was detected predominantly in a 100,000 x g pellet, with a low level of detection in a 9,000 x g pellet. Molecular sizes are indicated on the right in kDa. Results are representative of three independent experiments.

 
Tissue Distribution of mLPCAT1 mRNA—The tissue distribution of mLpcat1 was analyzed by quantitative real-time RT-PCR. We found the highest level of mLpcat1 expression in the lung, followed by spleen and brain. Other tissues had very low expression levels of mLpcat1 (Fig. 2).

Subcellular Localization of FLAG- or EGFP-mLPCAT1—To facilitate immunocytochemical and Western blot analysis of mLPCAT1, we constructed a fusion protein of mLPCAT1 that contained the FLAG or EGFP epitope fused in-frame to the N terminus of mLPCAT1. FLAG- or EGFP-mLpcat1 were transfected into CHO-K1 cells, and the enzyme distribution was examined by confocal microscopy after 48 h. Both FLAG- and EGFP-mLPCAT1 exhibited significant enzyme activities (data not shown). Cells were stained for ER-Golgi, mitochondria, and lysosome using DiOC6(3), MitoTracker Red, and LysoTracker Red, respectively. Whereas the subcellular distribution pattern of FLAG-mLPCAT1 was similar to that of DiOC6(3), EGFP-mLPCAT1 was not co-localized with MitoTracker Red or LysoTracker Red, suggesting that the enzyme is present mainly in the ER and Golgi (Fig. 3A). To confirm the results of microscopic observations, CHO-K1 cells transiently transfected with FLAG-mLPCAT1 were homogenized, and different subcellular fractions were collected by centrifugation. When these fractions were analyzed by Western blot using the M2 anti-FLAG antibody (Fig. 3B), the enzyme was found mostly in a 100,000 x g pellet, consistent with the confocal microscopic data (Fig. 3A). FLAG-mLPCAT1 had an apparent molecular mass of 60 kDa, a value consistent with the molecular weight predicted from the open reading frame of mLPCAT1 (Fig. 3B). FLAG-mLPCAT1 was also detected in a 9,000 x g pellet. According to Western blot analysis using anti-calnexin antibody as an ER maker, this fraction contained ER protein to a small extent (Fig. 3B).

Kinetics of mLPCAT1 Expressed in CHO-K1 Cells—We next examined the acyltransferase activity of mLPCAT1 using 1-palmitoyl-LPC and palmitoyl-CoA. The reaction was linear for the first few minutes at 30 °C (data not shown). The pH optimum for the reaction was between 7.4 and 10, and the reaction did not require Ca2+ (data not shown). Src homology-reducing reagents also had no effect on enzymatic activity (data not shown). Kinetic analysis was carried out by measuring acyltransferase activity in the microsomal fraction (100,000 x g pellet) derived from vector- or mLPCAT1-transfected CHO-K1 cells, using increasing concentrations (0–200 µM) of palmitoyl-CoA in the presence of 50 µM [1-14C]palmitoyl-LPC, or increasing concentrations (0–100 µM) of 1-palmitoyl-LPC in the presence of 25 µM [1-14C]palmitoyl-CoA (Fig. 4, A and B). The apparent Km values of mLPCAT1 for palmitoyl-CoA and 1-palmitoyl-LPC were 3.0 and 2.3 µM, respectively. At higher concentrations of palmitoyl-CoA, enzymatic activity appeared to be inhibited, possibly due to a detergent effect. Thus, the mLPCAT1 cDNA, when expressed in CHO-K1 cells, conferred significant LPCAT activity, using palmitoyl-CoA and 1-palmitoyl-LPC as substrates.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4.
Kinetic properties of mLPCAT1 expressed in CHO-K1 cells. Acyltransferase activity of mLPCAT1 was measured using increasing concentrations of palmitoyl-CoA and 50 µM [1-14C]palmitoyl-LPC (A), and using increasing concentrations of 1-paimitoyl-LPC and 25 µM [1-14C]palmitoyl-CoA (B), in the presence of 1 µg of the microsomal fraction from vector (open circles)- or mLPCAT1 (closed circles)-transfected cells. The data represent the mean ± S.D. of triplicate measurements. The results are representative of three independent experiments.

 
Substrate Selectivity of mLPCAT1—Using [1-14C]palmitoyl-CoA as an acyl donor, we analyzed the substrate specificity of mLPCAT1 using a variety of lysophospholipid acceptors (Fig. 5A). The microsomal fraction from CHO-K1 cells transfected with mLPCAT1 had detectable LPCAT activity in the presence of 1-acyl-LPC (Fig. 5A). 1-O-Alkyl-LPC, 1-O-alkenyl-LPC, and 1-acyl-LPG also served as acyl acceptors (supplemental figure). There also appeared to be endogenous acyltransferase activity toward 1-acyl-LPA and 1-acyl-LPC in CHO-K1 cells (open bars in Fig. 5A).

Next, we examined the preference of mLPCAT1 for various acceptor acyl groups at the sn-1 position of LPC, using [1-14C]palmitoyl-CoA as an acyl donor. As seen in Fig. 5B, mLPCAT1 recognized medium to long chain LPCs (C14:0 > C16:0 > C18:1 > C12:0, C18:0) as substrates.

We then examined the acyl-CoA selectivity of mLPCAT1 using [1-14C]palmitoyl-LPC as an acceptor (Fig. 5C). mLP-CAT1 demonstrated a clear preference for medium to long chain and saturated fatty acyl-CoAs (C10:0 > C8:0, C12:0 > C6:0, C14:0, C16:0). In addition, there was significant enzymatic activity toward C18 and C22 unsaturated fatty acyl-CoAs (Fig. 5C and supplemental materials Fig. A). In contrast, there was poor activity using short chain (C2 to C4) saturated fatty acyl-CoAs. Note that CHO-K1 cells had significant endogenous LPCAT activity toward long chain unsaturated fatty acyl-CoAs (open bars in Fig. 5C). These results suggested that the mLPCAT1 gene encoded an LPC acyltransferase with a preference for saturated acyl-CoAs.

Presence of LPCAT1 in Rat Alveolar Type II Cells—The high expression level of Lpcat1 in the mouse lung (Fig. 2), and its catalytic activity in producing both dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), strongly suggested that the enzyme would be present in alveolar type II cells. We isolated alveolar type II cells from the rat lung, because standard procedures for their isolation have been established (34), and a significant number of cells could be obtained. As shown in supplemental Fig. B, rat alveolar type II cells have typical structures, including intracellular lamellar bodies. Lpcat1 was enriched in this cell type, as determined by both quantitative RT-PCR (Fig. 6A) and enzyme assays using 1-palmitoyl-LPC and palmitoyl-CoA as substrates (Fig. 6B). Alveolar macrophages, which are another major cellular component of the lung, were obtained by lung lavage. Whereas Lpcat1 was not expressed in these cells (Fig. 6A), they exhibited LPCAT activity (Fig. 6B), possibly due to other types of lysophospholipid acyltransferases.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Pulmonary surfactant is a mixture of lipids and surfactant proteins secreted from alveolar type II cells (for reviews, see Ref. 35 and 36). It provides a low surface tension at the air-liquid interface for efficient gas exchange and alveolar stability. Both genetic and acquired deficiencies in pulmonary surfactant cause severe respiratory failure, which is manifest as acute respiratory distress syndrome, pneumonia, or neonatal respiratory distress syndrome (37, 38). Surfactant lipids also serve as binding and entry molecules for non-enveloped adenoviruses during common respiratory infections (39). Therefore, the production of surfactant lipids, especially DPPC, is highly regulated in vivo. Whereas there has been extensive analyses of surfactant proteins A, B, C, and D (38, 40), very little information is available on the mechanisms of production of surfactant lipids. Extensive analysis of surfactant composition revealed that there is 40% DPPC, and other PC, and that it contains 10% PG (41). Essentially, three distinct biosynthetic pathways are proposed to contribute to the composition of surfactant: 1) de novo biosynthesis, 2) remodeling by LPC or LPG acyltransferases, and 3) CoA-independent transacylation with 2 molecules of LPC (1, 2, 42–48). In an attempt to understand the relative contribution of each of these three pathways, we have isolated an enzyme that is involved in the production of both PC and PG via the remodeling pathway. By searching the genomic data base using conserved motifs of LPA acyltransferases (27) and tafazzins as queries, we identified a cDNA that encoded a protein of 534 amino acid residues, and had a predicted molecular mass of 60 kDa (Figs. 1 and 3B). It contained three motifs, motif 1 (HSSYFD), motif 2 (GTLIQ/RYIR), and motif 3 (PEGTC), that are conserved among three species (Fig. 1), and are possibly involved in catalytic activity.4 Using a heterologous overexpression system, we found that the enzyme was predominantly located in the ER-Golgi complex (Fig. 3). Whereas the small amount of mLPCAT1 was found in a 9.000 x g pellet (Fig. 3B), this could be due to the cross-contamination of ER protein in this fraction. In fact, calnexin, an ER maker was observed to the some extent in this fraction (data not shown). Thus, although there appears to be ER-Golgi-specific localization, the exact location of the endogenous protein in native cells remains to be determined.


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5.
Substrate specificity of mLPCAT1 expressed in CHO-K1 cells. A, lysophospholipid acyltransferase assays were performed with 50 µM lysophospholipid (LPA, LPC, LPE, LPG, LPI, or LPS) and 25 µM [1-14C]palmitoyl-CoA in the presence of 1 µg of the microsomal fraction from vector (open bars)- or mLPCAT1 (closed bars)-transfected cells. Lysophospholipid acyltransferase activities were toward 1-acyl-LPC (LPC), 1-O-alkyl-LPC, 1-O-alkenyl-LPC, and 1-acyl-LPG (LPG). 1-O-alkenyl-LPC (heart), 1-O-alkenyl-LPE (heart), LPI (liver), and LPS (brain) were from bovine tissues. Other lysophospholipids contained palmitoyl group at the sn-1 position. B, acyl acceptor selectivity of mLPCAT1 was analyzed by incubating microsomal fractions with 25 µM [1-14C]palmitoyl-CoA and 50 µM LPC (hexanoyl (C6:0), octanoyl (C8:0), decanoyl (C10:0), lauroyl (C12:0), myristoyl (C14:0), palmitoyl (C16:0), stearoyl (C18:0), oleoyl (C18:1), arachidoyl (C20:0), 1-O-hexadecyl (C1-O-16:0), 1-O-octadecyl (C1-O-18:0), and plasmalogen (C1-O-alkenyl). mLPCAT1 recognized medium to long chain LPCs (C14:0 > C16:0 > C18:1 > C12:0, C18:0) as substrates. C, acyl donor selectivity of mLPCAT1 for acyl-CoAs was analyzed by incubating microsomal fractions with 50 µM [1-14C]palmitoyl-LPC and 25 µM acyl-CoA (acetyl-CoA (C2:0), propionyl-CoA (C3:0), butyryl-CoA (C4:0), hexanoyl-CoA (C6:0), octanoyl-CoA (C8:0), decanoyl-CoA (C10:0), lauroyl-CoA (C12:0), myristoyl-CoA (C14: 0), palmitoyl-CoA (C16:0), stearoyl-CoA (C18:0), oleoyl-CoA (C18:1), linoleoyl-CoA (C18:2), {alpha}-linolenoyl-CoA (C18:3 n3), arachidoyl-CoA (C20:0), arachidonoyl-CoA (C20:4), and docosahexaenoyl-CoA (C22:6)). The data represents the mean ± S.D. of triplicate measurements. The results are representative of two independent experiments.

 


Figure 6
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 6.
Existence of LPCAT1 in rat alveolar type II cells. The expression levels of Lpcat1 and surfactant protein-C (Sp-C) were analyzed by quantitative real-time RT-PCR in rat lung, alveolar type II cells, and alveolar macrophages (A). The LPCAT activities in microsomal fractions from lung (n = 3), alveolar type II cells (n = 3), and alveolar macrophages (n = 2) were measured (B). Surfactant protein-C was used as a marker gene for alveolar type II cells. The data represents the mean ± S.E. of triplicate measurements.

 

The protein was highly expressed in alveolar type II cells of the rat lung (Fig. 6), and exhibited significant LPCAT activity, with a preference for medium chain (C6 to 14) saturated and long chain unsaturated fatty acyl-CoAs (Fig. 5C) in vitro. However, in alveolar type II cells, palmitoyl-CoA constitutes about 40% of all acyl-CoAs, and the medium chain acyl-CoAs are rarely seen in vivo (49). This is significant, because substrate availability determines the molecular species of PC in surfactant. It is interesting to note that mLPCAT1 also used LPG as an acceptor (Fig. 5A), because pulmonary surfactant contains PG, in addition to DPPC (41). Elucidation of the roles of mLPCAT1 in the production of components of surfactant, and in other biological processes awaits gene-targeting studies. It is also important to note that CHO-K1 cells have endogenous LPCAT activity, especially for long chain unsaturated fatty acyl-CoA donors (open bars in Fig. 5C). The identity of the endogenous enzyme(s) responsible for this activity is unknown, but these results point to enzymes involved in the deacylation and reacylation cycle (Lands' cycle) that occurs in various biological membranes (2–4). Further studies are needed to elucidate the roles of mLPCAT1 in vivo, and determine its potential as a therapeutic target for diseases involving surfactant dysfunction. Finally, the molecular identification and characterization of the first LPCAT paves the way for a better understanding of the biochemical mechanism underlying phospholipid biosynthesis.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Culture, Sports and Technology of Japan (to T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A and B.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB244717 [GenBank] (mouse), AB244719 [GenBank] (human), and AB244983 [GenBank] (rat). Back

1 Supported by the Center for NanoBio Integration at The University of Tokyo. Back

2 Supported by the Center for NanoBio Integration at The University of Tokyo. To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: tshimizu{at}m.u-tokyo.ac.jp.

3 The abbreviations used are: PC, phosphatidylcholine; BLAST, basic local alignment search tool; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; LPAAT, acyl-CoA:lysophosphatidic acid acyltransferase; LPCAT, acyl-CoA:lysophosphatidylcholine acyltransferase; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine; RT, reverse transcription; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; EGFP, epidermal growth factor protein; PG, phosphatidylglycerol; DPPC, dipalmitoylphosphatidylcholine. Back

4 T. Harayama, H. Shindou, and T. Shimizu, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Drs. A. Yamashita and K. Waku (Teikyo University) for valuable suggestions. We also thank Dr. J.-i. Miyazaki at Osaka University for supplying pCXN2. We acknowledge Drs. Yasuo Yoshida, Koujiro Tohyama, and Yoh-ichi Satoh for technical assistance and advice on alveolar type II cell morphology (Iwate Medical University). We also thank Drs. T. Yokomizo, S. Ishii, and all members of our laboratories for discussion, and T. Ohto and F. Hamano for technical assistance (The University of Tokyo).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kennedy, E. P. (1961) Fed. Proc. 20, 934–940[Medline] [Order article via Infotrieve]
  2. Lands, W. E. (1960) J. Biol. Chem. 235, 2233–2237[Free Full Text]
  3. Lands, W. E., and Merkl, I. (1963) J. Biol. Chem. 238, 898–904[Free Full Text]
  4. Merkl, I., and Lands, W. E. (1963) J. Biol. Chem. 238, 905–906[Free Full Text]
  5. Kume, K., and Shimizu, T. (1997) Biochem. Biophys. Res. Commun. 237, 663–666[CrossRef][Medline] [Order article via Infotrieve]
  6. Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856–10861[Abstract/Free Full Text]
  7. Green, P. R., Vanaman, T. C., Modrich, P., and Bell, R. M. (1983) J. Biol. Chem. 258, 10862–10866[Abstract/Free Full Text]
  8. Shin, D. H., Paulauskis, J. D., Moustaid, N., and Sul, H. S. (1991) J. Biol. Chem. 266, 23834–23839[Abstract/Free Full Text]
  9. Coleman, J. (1992) Mol. Gen. Genet. 232, 295–303[Medline] [Order article via Infotrieve]
  10. Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156–22163[Abstract/Free Full Text]
  11. Yet, S. F., Lee, S., Hahm, Y. T., and Sul, H. S. (1993) Biochemistry 32, 9486–9491[CrossRef][Medline] [Order article via Infotrieve]
  12. Eberhardt, C., Gray, P. W., and Tjoelker, L. W. (1997) J. Biol. Chem. 272, 20299–20305[Abstract/Free Full Text]
  13. West, J., Tompkins, C. K., Balantac, N., Nudelman, E., Meengs, B., White, T., Bursten, S., Coleman, J., Kumar, A., Singer, J. W., and Leung, D. W. (1997) DNA Cell Biol. 16, 691–701[Medline] [Order article via Infotrieve]
  14. Stamps, A. C., Elmore, M. A., Hill, M. E., Kelly, K., Makda, A. A., and Finnen, M. J. (1997) Biochem. J. 326, 455–461
  15. Aguado, B., and Campbell, R. D. (1997) Biochem. Soc. Trans. 25, S597[Medline] [Order article via Infotrieve]
  16. Aguado, B., and Campbell, R. D. (1998) J. Biol. Chem. 273, 4096–4105[Abstract/Free Full Text]
  17. Dircks, L., and Sul, H. S. (1999) Prog. Lipid Res. 38, 461–479[CrossRef][Medline] [Order article via Infotrieve]
  18. Li, D., Yu, L., Wu, H., Shan, Y., Guo, J., Dang, Y., Wei, Y., and Zhao, S. (2003) J. Hum. Genet. 48, 438–442[CrossRef][Medline] [Order article via Infotrieve]
  19. Lewin, T. M., Schwerbrock, N. M., Lee, D. P., and Coleman, R. A. (2004) J. Biol. Chem. 279, 13488–13495[Abstract/Free Full Text]
  20. Cao, J., Liu, Y., Lockwood, J., Burn, P., and Shi, Y. (2004) J. Biol. Chem. 279, 31727–31734[Abstract/Free Full Text]
  21. Yang, Y., Cao, J., and Shi, Y. (2004) J. Biol. Chem. 279, 55866–55874[Abstract/Free Full Text]
  22. Lu, B., Jiang, Y. J., Zhou, Y., Xu, F. Y., Hatch, G. M., and Choy, P. C. (2005) Biochem. J. 385, 469–477[CrossRef][Medline] [Order article via Infotrieve]
  23. Testet, E., Laroche-Traineau, J., Noubhani, A., Coulon, D., Bunoust, O., Camougrand, N., Manon, S., Lessire, R., and Bessoule, J. J. (2005) Biochem. J. 387, 617–626[CrossRef][Medline] [Order article via Infotrieve]
  24. Ye, G. M., Chen, C., Huang, S., Han, D. D., Guo, J. H., Wan, B., and Yu, L. (2005) DNA Seq. 16, 386–390[Medline] [Order article via Infotrieve]
  25. Barth, P. G., Wanders, R. J., Vreken, P., Janssen, E. A., Lam, J., and Baas, F. (1999) J. Inherit. Metab. Dis. 22, 555–567[CrossRef][Medline] [Order article via Infotrieve]
  26. Yamashita, A., Kawagishi, N., Miyashita, T., Nagatsuka, T., Sugiura, T., Kume, K., Shimizu, T., and Waku, K. (2001) J. Biol. Chem. 276, 26745–26752[Abstract/Free Full Text]
  27. Lewin, T. M., Wang, P., and Coleman, R. A. (1999) Biochemistry 38, 5764–5771[CrossRef][Medline] [Order article via Infotrieve]
  28. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193–199[CrossRef][Medline] [Order article via Infotrieve]
  29. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  30. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911–917
  31. Dobbs, L. G., and Mason, R. J. (1979) J. Clin. Investig. 63, 378–387[Medline] [Order article via Infotrieve]
  32. Shikano, S., and Li, M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5783–5788[Abstract/Free Full Text]
  33. Maki, M., Kitaura, Y., Satoh, H., Ohkouchi, S., and Shibata, H. (2002) Biochim. Biophys. Acta 1600, 51–60[Medline] [Order article via Infotrieve]
  34. Mason, R. J., and Dobbs, L. G. (1980) J. Biol. Chem. 255, 5101–5107[Abstract/Free Full Text]
  35. Veldhuizen, R., Nag, K., Orgeig, S., and Possmayer, F. (1998) Biochim. Biophys. Acta 1408, 90–108[Medline] [Order article via Infotrieve]
  36. Halliday, H. L. (2005) Biol. Neonate 87, 317–322[CrossRef][Medline] [Order article via Infotrieve]
  37. Lewis, J. F., and Jobe, A. H. (1993) Am. Rev. Respir. Dis. 147, 218–233[Medline] [Order article via Infotrieve]
  38. Floros, J., and Kala, P. (1998) Annu. Rev. Physiol. 60, 365–384[CrossRef][Medline] [Order article via Infotrieve]
  39. Balakireva, L., Schoehn, G., Thouvenin, E., and Chroboczek, J. (2003) J. Virol. 77, 4858–4866[Abstract/Free Full Text]
  40. Suwabe, A., Mason, R. J., Smith, D., Firestone, J. A., Browning, M. D., and Voelker, D. R. (1992) J. Biol. Chem. 267, 19884–19890[Abstract/Free Full Text]
  41. Yu, S. H., and Possmayer, F. (2003) J. Lipid Res. 44, 621–629[Abstract/Free Full Text]
  42. Yamada, K., and Okuyama, H. (1979) Arch. Biochem. Biophys. 196, 209–219[CrossRef][Medline] [Order article via Infotrieve]
  43. van Heusden, G. P., Vianen, G. M., and van den Bosch, H. (1980) J. Biol. Chem. 255, 9312–9318[Free Full Text]
  44. Funkhouser, J. D., Batenburg, J. J., and Van Golde, L. M. (1981) Biochim. Biophys. Acta 666, 1–6[Medline] [Order article via Infotrieve]
  45. Van Heusden, G. P., and Van den Bosch, H. (1981) Biochim. Biophys. Acta 666, 508–511[Medline] [Order article via Infotrieve]
  46. Okuyama, H., Yamada, K., Miyagawa, T., Suzuki, M., Prasad, R., and Lands, W. E. (1983) Arch. Biochem. Biophys. 221, 99–107[CrossRef][Medline] [Order article via Infotrieve]
  47. de Vries, A. C., Batenburg, J. J., and van Golde, L. M. (1985) Biochim. Biophys. Acta 833, 93–99[Medline] [Order article via Infotrieve]
  48. Yamashita, A., Sugiura, T., and Waku, K. (1997) J. Biochem. (Tokyo) 122, 1–16[Abstract/Free Full Text]
  49. den Breejen, J. N., Batenburg, J. J., and van Golde, L. M. (1989) Biochim. Biophys. Acta 1002, 277–282[Medline] [Order article via Infotrieve]
  50. Tusnady, G. E., and Simon, I. (2001) Bioinformatics 17, 849–850[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. Cao, D. Shan, T. Revett, D. Li, L. Wu, W. Liu, J. F. Tobin, and R. E. Gimeno
Molecular Identification of a Novel Mammalian Brain Isoform of Acyl-CoA:Lysophospholipid Acyltransferase with Prominent Ethanolamine Lysophospholipid Acylating Activity, LPEAT2
J. Biol. Chem., July 4, 2008; 283(27): 19049 - 19057.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Harayama, H. Shindou, R. Ogasawara, A. Suwabe, and T. Shimizu
Identification of a Novel Noninflammatory Biosynthetic Pathway of Platelet-activating Factor
J. Biol. Chem., April 25, 2008; 283(17): 11097 - 11106.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Q. Chen, M.-S. Kuo, S. Li, H. H. Bui, D. A. Peake, P. E. Sanders, S. J. Thibodeaux, S. Chu, Y.-W. Qian, Y. Zhao, et al.
AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase
J. Biol. Chem., April 11, 2008; 283(15): 10048 - 10057.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Zhao, Y.-Q. Chen, T. M. Bonacci, D. S. Bredt, S. Li, W. R. Bensch, D. E. Moller, M. Kowala, R. J. Konrad, and G. Cao
Identification and Characterization of a Major Liver Lysophosphatidylcholine Acyltransferase
J. Biol. Chem., March 28, 2008; 283(13): 8258 - 8265.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
H.-C. Lee, T. Inoue, R. Imae, N. Kono, S. Shirae, S. Matsuda, K. Gengyo-Ando, S. Mitani, and H. Arai
Caenorhabditis elegans mboa-7, a Member of the MBOAT Family, Is Required for Selective Incorporation of Polyunsaturated Fatty Acids into Phosphatidylinositol
Mol. Biol. Cell, March 1, 2008; 19(3): 1174 - 1184.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. Hishikawa, H. Shindou, S. Kobayashi, H. Nakanishi, R. Taguchi, and T. Shimizu
Discovery of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity
PNAS, February 26, 2008; 105(8): 2830 - 2835.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
E. Soupene, H. Fyrst, and F. A. Kuypers
Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes
PNAS, January 8, 2008; 105(1): 88 - 93.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Tamaki, A. Shimada, Y. Ito, M. Ohya, J. Takase, M. Miyashita, H. Miyagawa, H. Nozaki, R. Nakayama, and H. Kumagai
LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae
J. Biol. Chem., November 23, 2007; 282(47): 34288 - 34298.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Jain, N. Stanford, N. Bhagwat, B. Seiler, M. Costanzo, C. Boone, and P. Oelkers
Identification of a Novel Lysophospholipid Acyltransferase in Saccharomyces cerevisiae
J. Biol. Chem., October 19, 2007; 282(42): 30562 - 30569.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. R. Riekhof, J. Wu, J. L. Jones, and D. R. Voelker
Identification and Characterization of the Major Lysophosphatidylethanolamine Acyltransferase in Saccharomyces cerevisiae
J. Biol. Chem., September 28, 2007; 282(39): 28344 - 28352.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
A. K Agarwal, S. Sukumaran, R. Bartz, R. I Barnes, and A. Garg
Functional characterization of human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 9: cloning, tissue distribution, gene structure, and enzymatic activity
J. Endocrinol., June 1, 2007; 193(3): 445 - 457.
[Abstract] [Full Text] [PDF]