J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M400364-JLR200 on February 1, 2005

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M400364-JLR200v1
46/4/727    most recent
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 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 Liu, D.
Right arrow Articles by Zoeller, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, D.
Right arrow Articles by Zoeller, R. A.
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?
Journal of Lipid Research, Vol. 46, 727-735, April 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids

Dailan Liu*, Narasimhan Nagan1,*, Wilhelm W. Just{dagger}, Claus Rodemer{dagger}, Thanh-Phuong Thai{dagger} and Raphael A. Zoeller2,*

* Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118
{dagger} Biochemie-Zentrum, Universitat Heidelberg, D-69120 Heidelberg, Germany

Published, JLR Papers in Press, February 1, 2005. DOI 10.1194/jlr.M400364-JLR200

1 Present address of N. Nagan: Athena Diagnostics, Inc., Four Biotech Park, 377 Plantation Street, Worcester, MA 01605. Back

2 To whom correspondence should be addressed. e-mail: rzoeller{at}bu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273–279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis.

These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells.

Abbreviations: DHAP, dihydroxyacetonephosphate; DHAPAT, dihydroxyacetonephosphate acyltransferase; Gro-3-P, glycerol-3-phosphate; Gro-3-PAT, glycerol-3-phosphate acyltransferase; Pi, inorganic phosphate; PTS1, peroxisome targeting signal type 1; SSC, saline-sodium citrate buffer

Supplementary key words glycerol-3-phosphate acyltransferase • variant • Chinese hamster ovary cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmalogens represent a subset of phospholipids that differ from other glycerolipids in that, attached to the sn-1 position of the glycerol backbone is an alkyl chain attached through a vinyl ether linkage (1). The vinyl ether gives plasmalogens their unique physical and chemical properties. Every mammalian cell contains plasmalogens. In most cells, plasmalogens are represented as a subclass of ethanolamine phospholipids, in which they can represent a major portion of that head group class (2). Typically less abundant are choline plasmalogens, although in certain tissues, such as heart and muscle, they are found at relatively high levels. Plasmalogens have been proposed to be important for the stimulated release of arachidonic acid from phospholipid pools (3–5), the fusion of biological membranes (6), and the protection against reactive oxygen species (7–10).

The mechanism for the maintenance of plasmalogen levels and the rate-limiting factor(s) for plasmalogen biosynthesis have not been identified. Although the pathway for plasmenylethanolamine biosynthesis has been established, the exact biosynthetic pathway for plasmenylcholine has not. Unlike plasmenylethanolamine, the saturated ether lipid species, plasmanylcholine is not a precursor for plasmenylcholine (11). Plasmenylcholine appears to be synthesized from plasmenylethanolamine through the replacement of ethanolamine with choline (11, 12) or possibly through the methylation of plasmenylethanolamine (13). Our lack of knowledge in this area is attributable, in part, to the lack of molecular tools for studying the system. Only recently have any of the enzymes that participate in the biosynthetic pathway for plasmalogens been purified or the genes that code for them cloned (14, 15). Although early steps in any biosynthetic pathway are commonly candidates as control points, this paradigm may not apply to plasmalogen biosynthesis. The regulation of plasmalogen biosynthesis is likely to be complex and linked to the biosynthesis of nonether glycerolipids, because later steps in the pathway are also used in the synthesis of diacyl phospholipids and triglycerides (1). Recent evidence also suggests that dihydroxyacetonephosphate acyltransferase (DHAPAT), which catalyzes the first step in plasmalogen biosynthesis, may play a significant role in nonether glycerolipid biosynthesis as well; this activity is increased several-fold during the differentiation of 3T3-L1 cells to adipocytes (16). Therefore, the role of DHAPAT in the synthesis of this latter group of glycerolipids (nonether) remains to be determined.

We have described the isolation of variants of the fibroblast-like cell line CHO-K1 that are deficient in plasmalogens as a result of the loss of various steps along the biosynthetic pathway (17–19). These cell lines can be useful in identifying the factors that determine plasmalogen levels as well as in defining the relationship between ether lipids and diacyl lipid synthesis. The mutant CHO cell line NRel-4 displays greatly reduced plasmalogen levels because of a loss of DHAPAT (19). In this report, we show that the loss of this activity is attributable to reduced levels of the message for DHAPAT, making NRel-4 a useful null cell model for examining the role of DHAPAT in the regulation of plasmalogen levels. We have expressed the human DHAPAT gene in NRel-4 cells to demonstrate that this can recover plasmalogen biosynthesis in these cells and to examine what effect varied levels of DHAPAT activity have on the synthesis of both ether and nonether glycerolipids.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
[1-3H]ethanolamine (30 Ci/mmol; 1 Ci = 37 gigabecquerel) was obtained from Amersham (Arlington Heights, IL). [{gamma}-32P]ATP, [32P]inorganic phosphate (32Pi), [9,10-3H]oleic acid, and EN3 HANCE spray were obtained from Perkin-Elmer/New England Nuclear (Boston, MA). 32P-labeled dihydroxyacetonephosphate (DHAP) and glycerol-3-phosphate (Gro-3-P) were synthesized by enzymatic phosphorylation of dihydroxyacetone or glycerol using [{gamma}-32P]ATP and glycerol kinase (20). Ecoscint A liquid scintillation fluid was obtained from National Diagnostics (Atlanta, GA). Silica gel 60 TLC plates (Merck) were purchased from American Scientific Products (McGaw Park, IL). Tissue culture dishes (Corning) were obtained from VWR Scientific (Boston, MA). All other reagents, unless specified, were purchased from Sigma (St. Louis, MO).

Cell lines and culture conditions
CHO-K1 cells were obtained from the American Type Culture Collection (Manassas, VA). Plasmenylethanolamine is the dominant plasmalogen species, constituting ~35–40% of the ethanolamine phospholipids; there is almost no plasmenylcholine. NRel-4 is a plasmalogen-deficient CHO-K1-derived cell line with a severe deficiency in DHAPAT activity (19). NZel-1 is a plasmalogen-deficient variant of CHO-K1 defective in alkyl-DHAP synthase (18). Cells were grown in Ham's F12 medium supplemented with 10% fetal bovine serum (Atlas Biologicals, Denver, CO) at 37°C in 5% CO2.

Construction of an expression vector containing human DHAPAT
The human DHAPAT cDNA (14) was removed from the pBluescript SK phagemid vector and inserted into the shuttle vector pBK-CMV (Stratagene, La Jolla, CA) using the EcoRI (5') and XhoI (3') restriction sites. The construct was used to transform XL1-Blue MRF', which was then grown on kanamycin-containing agar to form colonies. Selected white colonies were expanded and cDNA preparations were analyzed by restriction analysis, and those strains whose cDNA demonstrated the appropriate agarose electrophoresis patterns were further expanded for large-scale DNA preparation using an endotoxin-free plasmid kit supplied by Qiagen (Valencia, CA). The cDNA inserts were sequenced to ensure that DHAPAT cDNA was unaltered and properly inserted. NRel-4 cells were transfected using SuperFect transfection reagent (Qiagen), and the transfected populations were grown for 6 weeks in G418 (500 µg/ml) to develop a stably transfected population.

To generate clonal isolates, a cell suspension was diluted to a concentration of 2 cells/ml, and 200 µl aliquots were transferred to the wells of a 96-well plate. After 10 days of growth at 37°C, wells were examined for the formation of a single colony. The cells in these wells were harvested with trypsin and expanded for further study. Conditioned medium (medium in which cells had been previously grown) was filtered using a sterile 0.22 µm filter and used for the dilutions and transfer to the 96-well plates. This was necessary to ensure that the cells would grow at very low density.

Enzyme assays
Glycerol-3-phosphate acyltransferase (Gro-3-PAT) and DHAPAT activities were measured using whole-cell homogenates that were prepared as described previously (18). DHAPAT and Gro-3-PAT activities were measured as described by Jones and Hajra (21) using palmitoyl-CoA and either 32P-labeled Gro-3-P or DHAP as the acyl acceptor. Protein content was determined using the method of Lowry et al. (22). Detection of DHAPAT in immobilized colonies (colony autoradiography) was performed as described previously (23). Briefly, transfected cells were plated onto a 100 mm diameter tissue culture dish at 300 cells per dish and allowed to attach overnight. Cells were then overlaid with a sterile polyester cloth. The cells were allowed to grow and form colonies within the polyester matrix for 8–10 days at 37°C. The polyester bearing the colonies was removed from the dish, washed once with PBS, and placed at –20°C overnight. The polyester cloth was thawed and placed in a 100 mm tissue culture dish containing 3 ml of DHAPAT assay mix at pH 5.5 (4 µCi [32P]DHAP/ml) and incubated for 15 min at 37°C. The reaction was stopped by the addition of 3 ml of ice-cold 20% TCA. The cloth was then placed on a Buchner funnel and rinsed five times with 25 ml portions of ice-cold 3% TCA to remove TCA-soluble substrate; the TCA-insoluble product, acyl-[32P]DHAP, remained associated with the colony. After drying for 1 h, the cloth was exposed to X-ray film overnight at –80°C. To visualize the colonies, the cloth was placed in 0.5% (w/v) Coomassie blue in methanol-water-acetic acid (45:45:10, v/v) for 30 min followed by destaining with methanol-water-acetic acid (45:45:10, v/v).

Ethanolamine labeling of cellular phospholipid
Cells (2.5 x 105) were grown for 18 h in sterile glass scintillation vials at 37°C in growth medium containing [1-3H]ethanolamine (1 µCi/ml). The medium was removed, the cells were washed once with 2 ml of PBS, and the lipids were extracted in 3.8 ml of CHCl3/methanol/PBS (1:2:0.8) containing 300 µg of a carrier lipid (beef heart lipids). After transferring to a test tube, 1 ml of CHCl3 and 1 ml of PBS were added to form a two-phase Bligh and Dyer system (24), and the lower (organic) phase was collected after centrifugation. Solvent was removed using a stream of nitrogen, and the labeled phospholipids were separated by two-stage, single-dimension TLC (23). The labeled species were visualized by exposure to X-ray film at –80°C after spraying the plates with EN3HANCE. Labeled bands were scraped into scintillation vials containing 1 ml of methanol, 8 ml of scintillation fluid was added, and radioactivity was quantitated by liquid scintillation spectrometry.

Northern analysis
A 454 bp fragment of the 5' end of CHO-K1 DHAPAT was generated by polymerase chain reaction using primers 5'-ATGGACGTTCCTAGCTCCTN-3' for the 5' end and 5'-AGCAGGACTACAGGGTGCTC-3' for the 3' end. The primers were designed based on the nucleotide sequence of the CHO-K1 DHAPAT gene (D. Liu, unpublished data). Probe (100 ng) and 50 ng of pTRI-B-Actin-Mouse (Ambion) were labeled with [{alpha}-32P]ATP using the RadPrime kit (Invitrogen, Carlsbad, CA). Total RNA was isolated from cells using Trizol reagent (Invitrogen). RNA was fractionated using a denaturing 0.8% agarose-formaldehyde gel, transferred to nylon membranes (Schleicher and Schuell, Keene, NH), air-dried, and fixed by ultraviolet cross-linking at 1,200 joules. Nylon membranes were prehybridized in Church-Gilbert hybridization buffer (0.5 M sodium phosphate, pH 7.4, 7% sodium dodecylsulfate, 1% BSA, and 1.0 mM EDTA) for 4 h at 62°C. The radioactive probes were denatured by boiling for 5 min and added to the hybridization buffer. After overnight hybridization at 62°C, the blots were washed sequentially as follows: 2x saline-sodium citrate buffer [1x saline-sodium citrate buffer (SSC) = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0] containing 0.5% SDS for 15 min at 42°C, 0.5x SSC and 0.5% SDS at 42°C for 15 min, and 0.1x SSC and 0.5% SDS for 30 min at 42°C. The blots were then exposed to Kodak X-AR film at –80°C for 2 days.

Western analysis
Immunoblotting using the anti-DHAPAT antibody was carried out as described previously (14). Briefly, confluent monolayers were scraped and pelleted, solubilized in Solu-CHAPS (1 mM dithiothreitol, 1 mM EDTA, 15 mM CHAPS, 50 mM Tris-HCl, and 150 mM NaCl, pH 7.0), and proteins were precipitated using 10% TCA. Proteins were separated using 12% SDS-PAGE and subsequently transblotted into polyvinylidene difluoride membranes. After blocking with PBS and 5% nonfat dry milk, blots were processed with anti-DHAPAT antibody. Horseradish peroxidase-conjugated secondary antibody and chemiluminescence (ECL kit; Amersham, San Francisco, CA) were used to visualize the bands.

Subcellular localization of catalase and DHAPAT
Immunofluorescence microscopy was carried out as described previously (25). Briefly, cells were grown on glass coverslips, washed, and fixed with 3% paraformaldehyde. After 5 min of permeabilization in 1% Triton X-100, cells were washed and incubated for 1 h with anti-catalase (25) and anti-DHAPAT (14) antibodies that had been raised in rabbits. After washing, secondary goat anti-rabbit antibodies (Sigma) coupled to either FITC (for DHAPAT) or tetramethyl rhodamine isothiocyanate (for catalase) were used to detect the proteins. Cells were then mounted on slides using moviol/paraphenylendiamine, and images were taken with a Zeiss Axiovert microscope.

Phospholipid composition, biosynthesis, and turnover
Cells were plated into sterilized glass scintillation vials and grown for 72 h at 37°C in medium containing 32Pi (2.5 µCi/ml) to label the phospholipids to constant specific activity. The medium was removed, the cell monolayer was washed once with PBS, and cellular lipids were extracted as described above. Phospholipids were separated using two-dimensional TLC (26). Between dimensions, the region containing lipids was sprayed with 10 mM HgCl2 in glacial acetic acid to cleave the vinyl ether bond of the plasmenyl species, allowing for the separation of the resulting sn-1-lysophospholipid and the unaffected diacyl species (27). Plates were exposed to GBX-2 X-ray film at –80°C after preflash. 32Pi-labeled phospholipids were scraped from the TLC plates directly into scintillation vials for quantitation by liquid scintillation spectrometry. For short-term labeling, cells were incubated for 3 h in medium containing 32Pi (20 µCi/ml) and samples were processed as above.

To measure the turnover of total phospholipid pools, cells were labeled for 72 h with 32Pi (2.5 µCi/ml). Cells were harvested with trypsin and plated into sterile glass scintillation vials (105 cells/vial) in 1 ml of medium containing 32Pi (2.5 µCi/ml) and allowed to attach overnight. Labeling medium was removed and replaced with 2 ml of unlabeled medium. Cellular phospholipids were extracted at 0, 24, and 48 h after removal of the labeled medium. Solvent was removed by evaporation under a nitrogen stream, samples were resuspended in CHCl3, and an aliquot was transferred to a scintillation vial. After allowing the solvent to evaporate, 1 ml of methanol was added, followed by 8 ml of scintillation fluid, and the amount of chloroform-soluble radioactivity was determined using liquid scintillation spectrometry.

[9,10-3H]oleate labeling of cellular lipids
Cells were plated into sterilized glass scintillation vials and allowed to attach overnight. Medium was removed and replaced with 1 ml of growth medium containing 2 µM [9,10-3H]oleic acid. After 3 h at 37°C, medium was removed, the cell monolayer was washed once with 3 ml of growth medium, and the cellular lipids were extracted as described above. An aliquot of each sample was run on silica gel 60 using n-hexane-ethyl ether-acetic acid (70:30:1, v/v) to separate neutral lipids; another aliquot was separated on silica gel 60 using chloroform-methanol-acetic acid-water (25:15:3:1.5, v/v) to separate phospholipid head group classes. Plates were exposed to GBX-2 X-ray film at –80°C after spraying with EN3HANCE. Labeled lipids were scraped from the TLC plates directly into scintillation vials containing 1 ml of methanol followed by the addition of 8 ml of liquid scintillation cocktail. Radioactivity was quantitated by liquid scintillation spectrometry.

Analysis of subclass distribution of ethanolamine and choline phospholipids by analysis of diradyl species
To determine the relative levels of the different subclasses (diacyl, plasmanyl, and plasmenyl) within a head group class, lipids were extracted from unlabeled cells as above and isolated by development on silica gel G plates using chloroform-methanol-acetic acid-water (25:15:3:1.5, v/v). The choline and ethanolamine phospholipids were recovered from the plate and treated with phospholipase C followed by benzoylation of the resulting diradylglycerols (28). Diacyl, alkylacyl, and alkenylacyl benzoylated derivatives were separated by TLC (silica gel G) using benzene-hexane-ethyl ether (50:45:5, v/v), visualized by spraying the TLC plate with water, and recovered from the plate using chloroform-methanol (2:1). Solvent was removed by evaporation under a nitrogen stream, and samples were resuspended in 100% ethanol and quantitated by absorbance at 230 nm.

Statistical analysis
Multiple groups were analyzed by ANOVA, followed by Tukey's honestly significant difference multiple comparison procedure. The data in Table 2 were analyzed with Student's t-test.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Subclass distribution within the ethanolamine and choline phospholipids in CHO-K1 and isolate 4.15

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection and expression of DHAPAT in NRel-4 cells
The variant strain NRel-4, derived from the CHO-K1 cell line, is severely reduced in DHAPAT activity, resulting in a 90% decrease in plasmalogen levels (19). We inserted the human DHAPAT gene into the pBK-CMV vector downstream of the constitutive CMV promoter and used this to transfect NRel-4 cells. Analysis of the stably transfected population showed that DHAPAT activity and plasmenylethanolamine levels were partially recovered (Fig. 1) . When this population was screened, using colony autoradiography, for DHAPAT activity (Fig. 2) , ~15% of the colonies displayed a strong signal, indicating recovery of activity. A control population, transfected with pBK-CMV vector alone, displayed no increase in plasmalogen content, and we were unable to identify any DHAPAT-positive colonies by colony autoradiography (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1. Dihydroxyacetonephosphate acyltransferase (DHAPAT) activities and plasmenylethanolamine levels are partially recovered in an NRel-4 population transfected with DHAPAT cDNA. Cells were transfected with the pBK-CMV vector bearing the DHAPAT cDNA. Transfected populations were used after selection in G418 for 6 weeks. DHAPAT activity was measured in whole-cell homogenates at pH 5.5, as described in Materials and Methods. DHAPAT activity in CHO-K1 homogenates was 540 pmol/min/mg. For ethanolamine labeling, cells were labeled for 24 h with [1-3H]ethanolamine, cellular lipids were extracted, phosphatidylethanolamine and plasmenylethanolamine were separated using a two-stage, single-dimension TLC system, and radioactivity associated with each subclass was determined as described in Materials and Methods. Any plasmanylethanolamine that may be labeled was not separated from the phosphatidylethanolamine using this procedure. All values represent averages ± SD of three determinations within one representative experiment.

 


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 2. Colony autoradiography shows that a fraction of the transfectants are DHAPAT-positive. Cells were allowed to grow and form colonies within polyester cloth. DHAPAT assays were performed at pH 5.5, exposed to X-ray film, and then stained with Coomassie blue to visualize the colonies, as described in Materials and Methods. A: Coomassie blue-stained colonies. B: X-ray film showing DHAPAT activity associated with each colony. Horizontal arrows indicate DHAPAT-positive colonies, and vertical arrows indicate colonies displaying no DHAPAT activity.

 
Three clonal isolates, designated 4.1, 4.2, and 4.15, were generated from this transfected population. Whole-cell homogenates from these transfectants displayed varying amounts of DHAPAT activity when measured at pH 5.5, the optimal pH for DHAPAT (Table 1). Although isolates 4.1 and 4.2 had recovered only a small fraction of wild-type DHAPAT levels, isolate 4.15 displayed almost six times the level of DHAPAT activity compared with CHO-K1 cells.


View this table:
[in this window]
[in a new window]
  		TABLE 1. DHAPAT activities and ethanolamine phospholipid content in CHO strains

 
The phospholipid composition was determined using steady-state 32Pi labeling. Although plasmenylethanolamine, the predominant plasmalogen species in CHO cells, was reduced in NRel-4, levels were returned to normal in NRel-4.15 (Table 1). Isolates 4.1 and 4.2, which displayed only minor increases in DHAPAT activity, recovered plasmenylethanolamine to 45–55% of wild-type levels. With the exception of the ethanolamine subclass levels, there were no other significant changes in head group composition regardless of DHAPAT activity (data not shown).

The possibility existed that increased DHAPAT activity would result in the accumulation of the alkylacyl (plasmanyl) subclass. These are not separated from the diacyl subclass using the two-dimensional TLC system described above. Therefore, a more detailed analysis of the subclass distribution of the ethanolamine and choline phospholipids was performed (Table 2). Although plasmenylethanolamine (alkenylacyl) was the predominant ether-linked species in the ethanolamine phospholipids, there was little or none of this form in the choline phospholipids; plasmanylcholine (alkylacyl) was the dominant ether-linked species in this head group class. There was no increase in the amount of any ether-linked phospholipid species over wild-type levels in isolate 4.15.

Northern analysis (Fig. 3) using a probe directed to the 5' end of the message for CHO-K1 revealed a band in CHO-K1 corresponding to ~2 kb, the size of the published sequences from mouse (29) and human (14). This band is greatly reduced or missing in NRel-4. NRel-4.15 displayed a band with a somewhat higher molecular mass (as a result of the ß-galactosidase sequence associated with the initial transcript from the vector). Western analysis, using antibody against human DHAPAT, revealed a faint band of ~72 kDa in CHO-K1 and another plasmalogen-deficient CHO strain, NZel-1 (Fig. 4) . This was close to the known molecular mass (77 kDa) of DHAPAT. The antibody raised against the human protein did not cross-react well with the Chinese hamster protein, and it was impossible to determine if DHAPAT levels were reduced in NRel-4. Isolate 4.15, expressing the human DHAPAT, displayed a very strong signal that ran adjacent to these bands. Immunofluorescence microscopy (Fig. 5) showed that DHAPAT in isolate 4.15 colocalized with catalase, another peroxisomal protein.



View larger version (61K):
[in this window]
[in a new window]
  		Fig. 3. DHAPAT loss in NRel-4 is attributable to a reduction in mRNA levels. Total RNA was separated and probed for DHAPAT message as described in Materials and Methods. Northern analysis, using probes for ß-actin, gave similar signals in all lanes (not shown).

 


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4. Western blot analysis shows overexpression of DHAPAT in isolate 4.15. Proteins were loaded and separated on a 12% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. Blots were processed with anti-DAPAT antibody followed by horseradish peroxidase-conjugated secondary antibody and chemiluminescent reagents to visualize the bands, as described in Materials and Methods. NZel-1 is another plasmalogen-deficient variant of CHO-K1 with normal levels of DHAPAT activity (18). Plasmalogen loss in this cell line is attributable to a deficiency in alkyl-DHAP synthase.

 


View larger version (78K):
[in this window]
[in a new window]
  		Fig. 5. Immunofluorescence microscopy shows colocalization of DHAPAT with catalase in isolate 4.15. DHAPAT and catalase were visualized using antibody and secondary antibody labeled with FITC (DHAPAT) or tetramethyl rhodamine isothiocyanate (catalase), as described in Materials and Methods.

 
Measurement of phospholipid biosynthesis revealed a modest increase in the synthesis rates of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin, whereas phosphatidylinositol biosynthesis remained unchanged in NRel-4 cells compared with CHO-K1 cells (Table 3). The reduction of plasmenylethanolamine labeling was offset by an increase in labeling of the nonether phospholipid, phosphatidylethanolamine. Biosynthesis rates were returned to wild-type values in isolate 4.15 with the exception of phosphatidylinositol, which was somewhat depressed. Plasmenylethanolamine synthesis was also returned to nearly normal rates.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Short-term 32P labeling of cellular phospholipids

 
Overall phospholipid turnover was examined by monitoring the loss of label from phospholipid pools in cells that had been prelabeled with 32Pi as described previously (17). The loss of label was identical in CHO-K1 and NRel-4 cells, with the loss of ~50% of the label after 48 h (data not shown).

Results from labeling of the cellular lipids with [9,10-3H] oleic acid were somewhat different from those observed with 32Pi labeling (Table 4). There was increased labeling in only the ethanolamine phospholipid fraction in NRel-4 (versus CHO-K1); however, overexpression of DHAPAT in isolate 4.15 resulted in reduced uptake of oleate into all phospholipids except the phosphatidylinositol/phosphatidylserine fraction. As expected, labeling of alkyldiacylglycerols was dramatically reduced in NRel-4, and this was reversed in isolate 4.15. NRel-4 displayed increased labeling in the cholesterol ester fraction, with a return to wild-type levels in the transfectant. Triglyceride labeling was unaffected in NRel-4 but slightly reduced in the transfectant.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Short-term uptake of [9,10-3H]oleate into lipid fractions

 
DHAPAT activity can be measured over a very broad pH range (30). All three cell lines displayed DHAPAT activity when measured at pH 7.4 (Table 5). There was significant residual DHAPAT activity in mutant homogenates when measured at this pH, although this was reduced compared with values from CHO-K1. As expected, activity in the transfectant was increased ~4-fold over the wild-type activity. When Gro-3-PAT activity was measured, NRel-4 cells displayed a 60% increase in activity compared with CHO-K1 cells. Gro-3-PAT activity was returned to levels similar to those in CHO-K1 in isolate 4.15.


View this table:
[in this window]
[in a new window]
  		TABLE 5. DHAPAT and Gro-3-PAT activities in whole-cell homogenates at pH 7.4

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mammalian DHAPAT is a peroxisomal protein that is targeted to the peroxisome through the peroxisome targeting signal type 1 (PTS1) (31). A number of human genetic disorders have been described in which the primary lesion is a disruption in the targeting of peroxisomal proteins such as DHAPAT (32). This results in a loss of DHAPAT activity and decreased plasmalogen biosynthesis, as in NRel-4 cells. However, when we initially described the isolation of the NRel-4 cell line, we reported that alkyl-DHAP synthase activity (another peroxisomal protein), very long chain fatty acid (e.g., C26) levels, and phytanic acid oxidation were normal (19). Also, immunofluorescence microscopy showed catalase [which also uses the PTS1 system (33)] to be localized in discrete organelles, and digitonin-mediated release of catalase activity from the cells was latent. All of these data led us to the conclusion that NRel-4 cells have normal, functional peroxisomes and that the targeting peroxisomal systems were functional. The primary lesion in NRel-4 appeared to be a defect in the structural gene for DHAPAT. The data presented here confirm this. Northern analysis demonstrated a severe reduction in message for DHAPAT and transfection of NRel-4 with the human DHAPAT resulted in recovered DHAPAT activity, recovered plasmalogen biosynthesis, and colocalization of DHAPAT with catalase. The latter results also confirmed a functional PTS1 system. The NRel-4 cell line, therefore, can be used to examine the biosynthesis and functions of ether lipids without the complications associated with the loss of other peroxisomal functions.

The factors that regulate the production and levels of ether-linked phospholipids, including plasmalogens, in animal cells are not well understood. Previous studies argue that early steps in the pathway for plasmalogen biosynthesis are not involved in regulating plasmalogen levels; attempts to increase plasmalogen levels in both cultured cells (34) and animal tissues (35, 36) by media supplementation or feeding with alkylglycerols, which enter the biosynthetic pathway downstream of the first three steps in the pathway, failed to significantly increase plasmalogen levels, even though these compounds were incorporated into the plasmalogen pools. Recently, however, we demonstrated a 1.6- to 2-fold increase in plasmalogens in human endothelial cells when grown in alkylglycerol-supplemented medium (37). This was attributable, primarily, to a large increase in plasmenylcholine (although plasmenylethanolamine levels were also increased significantly). The question, therefore, remained of whether enzymes catalyzing the earlier steps in the pathway, such as DHAPAT, could be rate-limiting in the synthesis of plasmalogens.

The data presented here demonstrate that peroxisomal DHAPAT is absolutely required for plasmalogen biosynthesis. This is in agreement with evidence presented by Ofman, Lajmir, and Wanders (38) and Rodemer et al. (39). The evidence presented here also argues against this activity as a controlling factor in plasmalogen biosynthesis. First, the recovery of only a small amount of activity in isolates 4.1 and 4.2 resulted in substantial increases in plasmenylethanolamine content. These data agree with data from a DHAPAT-deficient variant of the murine macrophage cell line, 264.7. This variant strain, RAW.7, displayed 3.5% of parental DHAPAT activity, but plasmalogen levels were reduced by only 55% (40). Second, overexpression of peroxisomal DHAPAT activity in the NRel-4.15 transfectant did not result in increases in plasmalogen biosynthesis or increase their levels above normal. These results are in agreement with the observation that plasmalogen levels are not increased in differentiating 3T3-L1 cells, in which DHAPAT is induced several-fold (16).

Our findings here demonstrate that significant changes in peroxisomal DHAPAT activity do not influence the production of diacylated glycerophospholipids or triacylglycerols. Loss or overexpression of this activity had little or no effect on triacylglycerol production, whereas loss of DHAPAT activity actually resulted in a modest increase in glycerophospholipid biosynthesis. These findings appear to be in contrast with those of Hajra et al. (16), who reported induction of peroxisomal DHAPAT activity in differentiating 3T3-L1 cells. However, acyl/alkyl-DHAP reductase is also upregulated several-fold in these cells. It is possible that this latter activity, required for the reduction of the ketone at the sn-2 position and the formation of lysophosphatidic acid, is rate-limiting in animal cells and must also be induced for DHAPAT to contribute to the synthesis of nonether glycerolipids. The reductase may also be important for the transport of lysophosphatidic acid produced in the peroxisome to the endoplasmic reticulum for further metabolism.

It is not clear why Gro-3-PAT activity increased in NRel-4. It does not appear to be related to the loss of ether lipids; other ether lipid-deficient strains have not demonstrated a similar increase (18). We thought that it may be related to a loss of peroxisomal DHAPAT message, because recovery of peroxisomal DHAPAT activity in isolate 4.15 restored Gro-3-PAT activity to a wild-type-like level. However, of three additional, independently isolated CHO strains deficient in peroxisomal DHAPAT activity, all within the same complementation group as NRel-4 and all displaying greatly reduced message for peroxisomal DHAPAT (none detected), only one showed a significant increase in Gro-3-PAT (A. G. Karthik, unpublished observations).

In summary, we have shown that acyl-DHAP, produced by peroxisomal DHAPAT, is absolutely required for plasmalogen biosynthesis in animal cells and that this activity does not appear to be a limiting factor in plasmalogen biosynthesis in CHO cells. We have also shown that this activity does not contribute, independently of other changes, to the synthesis of diacyl glycerolipids.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health Grant GM-50571 and American Heart Association Grant 0455753T to R.A.Z.

Manuscript received September 23, 2004 and in revised form January 13, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Nagan, N., and R. A. Zoeller. 2001. Plasmalogens: biosynthesis and functions. Prog. Lipid Res. 40: 199–229.[CrossRef][Medline]

  2. Horrocks, L. A., and M. Sharma. 1982. Plasmalogens and O-alkylglycerophospholipids. In New Comprehensive Biochemistry. Neuberger, A. and L. L. M. van Deenen, editors. Elsevier Biomedical Press, Amsterdam. 51–95.

  3. Ford, D. A., and R. W. Gross. 1989. Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation. Proc. Natl. Acad. Sci. USA. 86: 3479–3483.[Abstract/Free Full Text]

  4. Yang, H. C., A. A. Farooqui, and L. A. Horrocks. 1996. Characterization of plasmalogen-selective phospholipase A2 from bovine brain. Adv. Exp. Med. Biol. 416: 309–313.[Medline]

  5. Creer, M. H., and J. McHowat. 1998. Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation. Am. J. Physiol. 275: C1498–C1507.

  6. Glaser, P. E., and R. W. Gross. 1994. Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adopt an HII phase with its ability to promote membrane fusion. Biochemistry. 33: 5805–5812.[CrossRef][Medline]

  7. Zoeller, R. A., O. H. Morand, and C. R. H. Raetz. 1988. A possible role for plasmalogens in protecting animal cells against photosensitized killing. J. Biol. Chem. 263: 11590–11596.[Abstract/Free Full Text]

  8. Morand, O. H., R. A. Zoeller, and C. R. H. Raetz. 1988. Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation. J. Biol. Chem. 263: 11597–11606.[Abstract/Free Full Text]

  9. Zoeller, R. A., A. C. Lake, N. Nagan, D. P. Gaposchkin, M. A. Legner, and W. Lieberthal. 1999. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether. Biochem. J. 338: 769–776.

  10. Engelmann, B., C. Brautigam, and J. Thiery. 1994. Plasmalogen phospholipids as potential protectors against lipid peroxidation of low density lipoproteins. Biochem. Biophys. Res. Commun. 204: 1235–1242.[CrossRef][Medline]

  11. Lee, T. C., C. G. Qian, and F. Snyder. 1991. Biosynthesis of choline plasmalogens in neonatal rat myocytes. Arch. Biochem. Biophys. 286: 498–503.[CrossRef][Medline]

  12. Ford, D. A., and R. W. Gross. 1988. Identification of endogenous 1-O-alk-1'-enyl-2-acyl-sn-glycerol in myocardium and its effective utilization by choline phosphotransferase. J. Biol. Chem. 263: 2644–2650.[Abstract/Free Full Text]

  13. Mozzi, R., D. Gramignani, C. Andriamampandr, L. Freysz, and R. Massarelli. 1989. Choline plasmalogen synthesis by the methylation pathway in chick neurons in culture. Neurochem. Res. 14: 579–583.[CrossRef][Medline]

  14. Thai, T. P., H. Heid, H. R. Rackwitz, A. Hunziker, K. Gorgas, and W. W. Just. 1997. Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase. FEBS Lett. 420: 205–211.[CrossRef][Medline]

  15. de Vet, E. C., A. W. Zomer, G. J. Lahaut, and H. van den Bosch. 1997. Polymerase chain reaction-based cloning of alkyl-dihydroxyacetonephosphate synthase complementary DNA from guinea pig liver. J. Biol. Chem. 272: 798–803.[Abstract/Free Full Text]

  16. Hajra, A. K., L. K. Larkins, A. K. Das, N. Hemati, R. L. Erickson, and O. A. MacDougald. 2000. Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis. J. Biol. Chem. 275: 9441–9446.[Abstract/Free Full Text]

  17. James, P. F., A. C. Lake, A. K. Hajra, L. K. Larkins, M. Robinson, F. G. Buchanan, and R. A. Zoeller. 1997. An animal cell mutant with a deficiency in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Effects on the biosynthesis of ether-linked and diacyl glycerolipids. J. Biol. Chem. 272: 23540–23546.[Abstract/Free Full Text]

  18. Nagan, N., A. K. Hajra, A. K. Das, H. W. Moser, A. Moser, P. Lazarow, P. E. Purdue, and R. A. Zoeller. 1997. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis. Proc. Natl. Acad. Sci. USA. 94: 4475–4480.[Abstract/Free Full Text]

  19. Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273–279.

  20. Schlossman, D. M., and R. M. Bell. 1977. Microsomal sn-glycerol 3-phosphate and dihydroxyacetone phosphate acyltransferase activities from liver and other tissues. Evidence for a single enzyme catalyzing both reactions. Arch. Biochem. Biophys. 182: 732–742.[CrossRef][Medline]

  21. Jones, C. L., and A. K. Hajra. 1980. Properties of guinea pig liver peroxisomal dihydroxyacetone phosphate acyltransferase. J. Biol. Chem. 255: 8289–8295.[Free Full Text]

  22. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275.[Free Full Text]

  23. Zoeller, R. A., and C. R. H. Raetz. 1992. Strategies for isolating somatic cell mutants defective in lipid biosynthesis. Methods Enzymol. 209: 34–51.[Medline]

  24. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911–917.

  25. Soto, U., R. Pepperkok, W. Ansorge, and W. W. Just. 1993. Import of firefly luciferase into mammalian peroxisomes in vivo requires nucleoside triphosphates. Exp. Cell Res. 205: 66–75.[CrossRef][Medline]

  26. Esko, J. D., and C. R. H. Raetz. 1980. Mutants of Chinese hamster ovary cells with altered membrane phospholipid composition. Replacement of phosphatidylinositol by phosphatidylglycerol in a myo-inositol auxotroph. J. Biol. Chem. 255: 4474–4480.[Free Full Text]

  27. Owens, K. 1966. A two-dimensional thin-layer chromatographic procedure for the estimation of plasmalogens. Biochem. J. 100: 354–361.[Medline]

  28. Blank, M. L., E. A. Cress, P. Lee, N. Stephens, C. Piantadosi, and F. Snyder. 1983. Quantitative analysis of ether-linked lipids as alkyl- and alk-1-enyl-glycerol benzoates by high-performance liquid chromatography. Anal. Biochem. 133: 430–436.[CrossRef][Medline]

  29. Thai, T. P., C. Rodemer, J. Worsch, A. Hunziker, K. Gorgas, and W. W. Just. 1999. Synthesis of plasmalogens in eye lens epithelial cells. FEBS Lett. 456: 263–268.[CrossRef][Medline]

  30. Webber, K. O., N. S. Datta, and A. K. Hajra. 1987. Properties of the enzymes catalyzing the biosynthesis of lysophosphatidate and its ether analog in cultured fibroblasts from Zellweger syndrome patients and normal controls. Arch. Biochem. Biophys. 254: 611–620.[CrossRef][Medline]

  31. Ofman, R., E. H. Hettema, E. M. Hogenhout, U. Caruso, A. O. Muijsers, and R. J. Wanders. 1998. Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2. Hum. Mol. Genet. 7: 847–853.[Abstract/Free Full Text]

  32. Brites, P., H. R. Waterham, and R. J. Wanders. 2004. Functions and biosynthesis of plasmalogens in health and disease. Biochim. Biophys. Acta. 1636: 219–231.[Medline]

  33. Purdue, P. E., and P. B. Lazarow. 1996. Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence. J. Cell Biol. 134: 849–862.[Abstract/Free Full Text]

  34. Cabot, M. C., and F. Snyder. 1980. Manipulation of alkylglycerolipid levels in cultured cells. Fatty alcohol versus alkylglycerol supplements. Biochim. Biophys. Acta. 617: 410–418.[Medline]

  35. Blank, M. L., E. A. Cress, Z. L. Smith, and F. Snyder. 1991. Dietary supplementation with ether-linked lipids and tissue lipid composition. Lipids. 26: 166–169.[Medline]

  36. Das, A. K., and A. K. Hajra. 1988. High incorporation of dietary 1-O-heptadecyl glycerol into tissue plasmalogens of young rats. FEBS Lett. 227: 187–190.[CrossRef][Medline]

  37. Zoeller, R. A., T. J. Grazia, P. LaCamera, J. Park, D. P. Gaposchkin, and H. W. Farber. 2002. Increasing plasmalogen levels protects human endothelial cells during hypoxia. Am. J. Physiol. 283: H671–H679.

  38. Ofman, R., S. Lajmir, and R. J. A. Wanders. 2001. Etherphospholipid biosynthesis and dihydroxyacetone-phosphate acyltransferase: resolution of the genomic organization of the GNPAT gene and its use in the identification of novel mutations. Biochem. Biophys. Res. Commun. 281: 754–760.[CrossRef][Medline]

  39. Rodemer, C., T. P. Thai, B. Brugger, T. Kaercher, H. Werner, K. A. Nave, F. Wieland, K. Gorgas, and W. W. Just. 2003. Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet. 12: 1881–1895.[Abstract/Free Full Text]

  40. Zoeller, R. A., S. Rangaswamy, H. Herscovitz, W. B. Rizzo, A. K. Hajra, A. K. Das, H. W. Moser, A. Moser, P. B. Lazarow, and M. J. Santos. 1992. Mutants in a macrophage-like cell line are defective in plasmalogen biosynthesis, but contain functional peroxisomes. J. Biol. Chem. 267: 8299–8306.[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
R. Zufferey and C. B. Mamoun
Leishmania major Expresses a Single Dihydroxyacetone Phosphate Acyltransferase Localized in the Glycosome, Important for Rapid Growth and Survival at High Cell Density and Essential for Virulence
J. Biol. Chem., March 24, 2006; 281(12): 7952 - 7959.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M400364-JLR200v1
46/4/727    most recent
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 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 Liu, D.
Right arrow Articles by Zoeller, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, D.
Right arrow Articles by Zoeller, R. A.
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?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today