From the Medical Research Council Immunochemistry Unit, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom
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ABSTRACT |
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Sequence analysis of cDNA clones
corresponding to a number of genes located in the class III region of
the human major histocompatibility complex (MHC), in the chromosome
band 6p21.3, has shown that the G15 gene encodes a
283-amino acid polypeptide with significant homology over the entire
polypeptide with the enzyme lysophosphatidic acid acyltransferase
(LPAAT) from different yeast, plant, and bacterial species. The amino
acid sequence of the MHC-encoded human LPAAT (hLPAAT) is 48%
identical to the recently described hLPAAT (Eberhardt, C., Gray,
P. W., and Tjoelker, L. W. (1997) J. Biol. Chem.
272, 20299-20305), which is encoded by a gene located on
chromosome 9p34.3. LPAAT is the enzyme that in lipid metabolism converts lysophosphatidic acid (LPA) into phosphatidic acid (PA). The
expression of the hLPAAT
polypeptide in the baculovirus system and
in mammalian cells has shown that it is an intracellular protein that
contains LPAAT activity. Cell extracts from insect cells overexpressing
hLPAAT
were analyzed in different LPAAT enzymatic assays using, as
substrates, different acyl acceptors and acyl donors. These cell
extracts were found to contain up to 5-fold more LPAAT activity
compared with control cell extracts, indicating that the hLPAAT
specifically converts LPA into PA, incorporating different acyl-CoAs
with different affinities. The hLPAAT
polypeptide expressed in the
mammalian Chinese hamster ovary cell line was found, by confocal
immunofluorescence, to be localized in the endoplasmic reticulum. Due
to the known role of LPA and PA in intracellular signaling and
inflammation, the hLPAAT
gene represents a candidate gene for some
MHC-associated diseases.
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INTRODUCTION |
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Lysophosphatidic acid acyltransferase (LPAAT),1 also known as 1-acyl-sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.51), is the enzyme that converts lysophosphatidic acid (LPA) into phosphatidic acid (PA) in the lipid metabolism. LPA or 1-acyl-sn-glycerol 3-phosphate consists of a glycerol backbone with a fatty acyl chain at the sn-1 position, a hydroxyl group at the sn-2 position, and a phosphate group at the sn-3 position. In the endoplasmic reticulum (ER) membrane, LPA is formed from glycerol 3-phosphate through the action of glycerol-3-phosphate acyltransferase. LPA is then further acylated in the ER by LPAAT to yield PA, the precursor of all glycerolipids. The rate of acylation of LPA to PA is very high, and consequently, there is little accumulation of LPA at the site of biosynthesis. PA can either be hydrolyzed by phosphatidic acid phosphohydrolase to yield diacylglycerol (DAG) or, alternatively, can be converted to CDP-DAG for the synthesis of more complex phospholipids in the ER, from which they are transported to different subcellular compartments. PA can be produced by this de novo synthesis or, alternatively, by phospholipase D hydrolysis of phospholipids, such as phospha- tidylcholine (PC) and phosphatidylethanolamine, or through phosphorylation of DAG by DAG kinase (for a general description, see Refs. 1 and 2).
Naturally occurring glycerolipids generally exhibit a nonrandom distribution of the acyl constituents; saturated fatty acids are esterified predominantly at the C-1 position and unsaturated fatty acids at the C-2 position (for a review see Refs. 3 and 4). Previous studies carried out using a semipurified LPAAT from rat liver microsomes indicated that LPAAT exhibited a significant acyl donor specificity for monoeic and dienoeic acyl-CoA thioesters (5). However, the activity of the enzyme was essentially not affected by the fatty acid constituent of the acyl acceptor (LPA), except that the 1-stearoyl and 1-arachidonoyl LPAs were somewhat less effective acyl acceptors (6). This acyl acceptor specificity was also found for lyso-PC acyltransferase when using different lysophosphatidylcholine (LPC) as substrates. The acyl acceptor specificity of LPAAT, however, does depend on the polar head group, and LPAAT is highly specific for LPA. In contrast, lyso-PC acyltransferase utilizes several acyl acceptors differing in the polar head group, except that LPA and lysophosphatidylethanol are ineffective substrates (6).
The human major histocompatibility complex (MHC) spans ~4 megabase pairs in the chromosome band 6p21.3 and is divided into three regions (7). The class I and II regions contain the classical MHC genes, which encode cell-surface glycoproteins involved in the presentation of antigenic peptides to T cells during an immune response. These are interspersed with a large number of other genes, some of which encode proteins involved in antigen processing. The class I and class II regions are separated by the central class III region that spans 1100 kilobase pairs of DNA (7, 8). Characterization of a 220-kilobase pair segment of DNA located between the class II region and the complement C4 genes in the class III region of the human MHC has revealed that the region contains at least nine genes (9-15), NOTCH-4, G18, PBX-2 (G17), RAGE, G16, G15, G14, G13 (Creb-rp), and TN-X (tenascin-X), of which only five (NOTCH-4, PBX-2, RAGE, Creb-rp, and TN-X), at present, encode proteins of known or putative function (for a review, see Ref. 8). We have found that one of the uncharacterized genes (G15) encodes a protein that has significant homology with LPAAT from bacteria, plant, and yeast species, suggesting that it could be the human homologue.
During the preparation of this manuscript, two other papers have been
published describing the cloning and expression of human LPAAT (hLPAAT)
(16, 17). In addition, another hLPAAT has been described (16, 18),
which is encoded by a gene located in chromosome 9 (18). For
convenience, West et al. (16) named the two hLPAATs as
LPAAT and LPAAT
. In this report, we describe the finding, through
cDNA sequence analysis and expression of the encoded polypeptide in
insect and mammalian cell lines, that the MHC class III region gene
G15 codes for a hLPAAT (hLPAAT
). We also report a
detailed characterization of the enzymatic activity of hLPAAT
and
the localization of the enzyme in the ER.
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EXPERIMENTAL PROCEDURES |
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cDNA Sequence Analysis-- Screening of a U937 cDNA library, using two overlapping cosmids (D3A and E91) from the MHC class III region as probes, resulted in the isolation of 22 cDNA clones (9). Characterization of these clones by restriction enzyme mapping revealed that pG15-3B contained a full-length cDNA insert of ~2.1 kilobase pairs. Both strands of this cDNA were sequenced by the dideoxy chain termination method after random sonicated fragments were cloned in the size range 300-1000 bp by blunt end ligation into SmaI-cut M13mp18. The sequence was assembled using the Staden (19) programs and determined with a degeneracy of 9.0. Computer analyses were performed using the software package of the University of Wisconsin Genetics Computer Group (GCG) (20).
Baculovirus Expression--
To remove the 3'- and 5'-flanking
sequences of the G15 cDNA (hLPAAT), to express only the coding
sequence under the control of the polyhedrin promoter, a PCR copy of
the open reading frame was generated using oligonucleotide primers that
also created XbaI sites adjacent to the initiating AUG codon
and the stop codon. The oligonucleotide primers used were as follows:
sense (5'-GCGGTCTAGAATGGATTTGTGGCCAGG-3') and
antisense (5'-CCGCTCTAGAGCCAGGGTTCACCCACC-3'), in
which the XbaI sites are in italics, and the initiating and
stop codons are in boldface type. This PCR copy was gel-isolated and
ligated into the XbaI-digested plasmid pBluescript
KS+ (pBlsc), and several clones were sequenced. The insert
of one clone (pG15Bls) that did not include any PCR errors was excised and ligated to the XbaI-cut baculovirus transfer vector
pAcCL29.1 (21), kindly donated by Dr. I. Jones (NERC Institute of
Virology and Environmental Microbiology, Oxford), to yield pG15Bac
(hLPAAT
).
Metabolic Labeling of Proteins-- Sf21 cells were infected at 10 plaque forming units/cell and pulse-labeled from 24 to 27 h postinfection with 200 µCi/ml Tran35S-label (ICN Biomedicals; a mixture of ~80% [35S]methionine and ~20% [35S]cysteine, 1200 Ci/mmol) in methionine-free TC100 medium in the absence of serum. Cells and extracellular media were analyzed by SDS-polyacrylamide gel electrophoresis in 12% acrylamide gels. Radioactive bands were detected by fluorography with Amplify (Amersham Corp.).
Subcloning of the cDNA Fragment into pcDNA3--
To
express the hLPAAT fused at the C terminus to a T7.Tag sequence
(MASMTGGQQMGRDP), for which specific monoclonal antibodies are
commercially available (T7.TagmAb) (Novagen), the last 270 bp encoding
the C terminus of the coding sequence of hLPAAT
(from pG15Bls) were
PCR-amplified. In this amplification, a NcoI site at the
3'-end of the cDNA was created to remove the stop codon and to fuse
hLPAAT
in frame to the T7.Tag sequence, located in T7.TagpBlsc
(kindly donated by C. Winchester, from this laboratory). The
oligonucleotide primers used for the PCR were as follows: sense
(5'-CCATCTTGCAGTGCAGGCC-3') and antisense
(5'-TAGCCATGGCACCGCCCCCAGGCTTCTTC-3'), in which the
NcoI site is indicated in italics and the disrupted stop
codon is shown in boldface type. The PCR product was isolated and
cloned into pBlsc, the DNA sequence was confirmed, and one clone
(pG15COOH-Blsc) was digested with AvaI-NcoI to
isolate the insert. To obtain the 5'-end of the coding sequence,
pG15Bls was digested with XbaI, end-filled, and then
digested with AvaI, and the 0.68-kilobase pair blunt
end/AvaI fragment was isolated and ligated together with the
AvaI-NcoI fragment encoding the C-terminal region into EcoRV/NcoI-digested T7.TagpBlsc. The
proper ligation of the two fragments into the vector was confirmed by
sequence analysis. A HindIII-XbaI insert,
containing the hLPAAT
cDNA fused to the T7.Tag sequence, was
isolated and cloned into pcDNA3 (Invitrogen) cut with
HindIII-XbaI to generate pcDNA3G15Tag.
Stable Expression in Chinese Hamster Ovary (CHO) Cells--
CHO
cells were electroporated with linearized (SspI)
pcDNA3G15Tag or pcDNA3 and incubated for 2 days with GMEMS (for
CHO cells, Advanced Protein Products Ltd.), supplemented with 10%
(v/v) fetal calf serum. After that time, cells were subcultured at
different densities containing different concentrations of geneticin
sulfate (G418). Expression of the protein was confirmed after 13 days of transfection by Western blot analysis (ECL method, Amersham) using
the T7.TagmAb, and those groups of cells expressing the hLPAAT
recombinant protein were diluted to obtain single clones expressing
hLPAAT
. Expression of the protein in the single clones was confirmed
by Western blotting, and three of them were analyzed by
immunofluorescence and for LPAAT activity. One of the clones, CHOG15
(hLPAAT
), was chosen for subsequent experiments. Single clones
containing only the vector were created in parallel, and the presence
of the vector was confirmed by PCR screening using specific pcDNA3
primers. Three of these clones were used in immunofluorescence and
enzyme activity assays as negative controls for the hLPAAT
transfectants, and one of these, CHOV, was chosen for further experiments.
Enzyme Assays--
Sf21 cells were infected with the wild
type (A. californica nuclear polyhedrosis virus) or vG15Bac
(hLPAAT) baculovirus at low multiplicity of infection (2 plaque-forming units/cell). Cells were harvested 72 h
postinfection (or when the cytopathic effect was total) and resuspended
in 50 mM Tris-HCl, pH 8, followed by four cycles of
freeze-thaw and then Dounce homogenization. The homogenates were spun
down for 10 min at 2000 rpm, and the supernatant was aliquoted and
stored at
70 °C.
Immunofluorescence-- Cells were grown on glass cover slides, fixed in 4% paraformaldehyde in 250 mM Hepes, pH 7.4, for 30 min, and quenched in 50 mM NH4Cl in PBS for 15 min at room temperature. The primary and secondary antibodies were added in 0.2% gelatin, 0.05% saponin in PBS for 45 min. The secondary antibody was fluorescein isothiocyanate-conjugated anti-mouse-IgG (Sigma Immunochemicals), while the 1D3 primary monoclonal antibody (mAb), was kindly donated by Dr D. Vaux (Sir William Dunn School of Pathology, Oxford). Nonpermeabilized conditions were without detergent and involved incubation of the primary mAb at 4 °C to avoid permeabilization and internalization of the mAbs and membrane proteins. Immunofluorescence was observed using a Bio-Rad MRC 1024 confocal microscope.
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RESULTS |
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The hLPAAT Gene Is Located in the MHC Class III Region--
A
nearly full-length cDNA clone (pG15-3B) corresponding to the
single copy gene G15, located in the MHC class III region, was isolated from a U937 cDNA library (9) and sequenced. The 2045-bp cDNA insert, with a poly(A) signal AATAAA 17 bp upstream of
a 21-bp poly(A) tail at one end (Fig.
1a), contained a single long
open reading frame, which was predicted to encode a 31.7-kDa protein.
The hydrophobicity plot (Fig. 1b) of the 283-amino acid predicted polypeptide (Fig. 1a) revealed the presence of
seven potential hydrophobic regions, suggesting that the G15
gene product could be a membrane-spanning protein. In addition, at the
N terminus there are two putative signal cleavage sites after amino
acids 22 and 58, respectively.
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Expression of hLPAAT in Insect Cells and in the Mammalian Cell
Line CHO--
To characterize the G15 gene product
(hLPAAT
), the protein was expressed in insect cells using the
baculovirus system. Radiolabeling of Sf21 insect cells infected
with vG15Bac (hLPAAT
) showed, in cell extracts, a major specific
polypeptide of 26 kDa and a minor specific polypeptide of 28 kDa that
were not observed in infections with a control virus expressing the
secreted vaccinia virus interleukin 1
(IL-1
) receptor (AcB15R)
(22) or with the wild type virus (Fig.
3a). hLPAAT
was not
detected in the medium (Fig. 3a), in contrast to the
secreted control AcB15R. When the radiolabeling was performed in the
presence of tunicamycin, no difference in the hLPAAT
size was
observed (data not shown), indicating that the single potential
N-glycosylation site situated at amino acids 184-186 (Fig.
1a) is not glycosylated, consistent with its predicted cytosolic localization (see below).
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Topology of the Protein--
The SigCleave program in the GCG
package predicts two signal peptides, one that would be cleaved after
amino acid 22 and the second after amino acid 58 (Fig. 1). Since the
expected molecular mass for hLPAAT is ~31.7 kDa, including the
signal peptide, the ~28- and ~26-kDa forms found in insect cells
could be explained by the processing of hLPAAT
after each of the
potential cleavage sites. However, in CHOG15 (hLPAAT
) cells, only a
single band of ~27 kDa was seen. When the molecular weight of the
14-amino acid T7.Tag is taken into account, the size of the hLPAAT
polypeptide will be ~26 kDa, and this is similar in size to the major
band found for hLPAAT
expressed in insect cells using baculovirus (Fig. 3). This suggests that the major cleavage site for the signal peptide is at amino acid 58 and that the cleavage at amino acid 22 observed in insect cells could be due to aberrant processing. Since it
is also possible that anomalous migration of the protein (due to its
hydrophobic nature) during SDS-polyacrylamide gel electrophoresis is
taking place, further experiments will be required to characterize the
amino terminus of the mature protein.
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1-Acylglycerol-3-Phosphate Acyltransferase Activity of
hLPAAT--
To demonstrate that the G15 gene product is
the hLPAAT
, the activity of the recombinant protein expressed using
the baculovirus system was characterized in vitro by
spectrophotometric and TLC analysis. To define the acyl-CoA (acyl
donor) specificity of hLPAAT
in relation to the length and degree of
saturation of the fatty acid, we have used different saturated
(stearoyl (C18:0), lignoceroyl (C24:0), arachidoyl (C20:0), palmitoyl
(C16:0), myristoyl (C14:0), and lauroyl (C12:0)) or unsaturated
(palmitoleoyl (C16:1), arachidonoyl (C20:4), linolenoyl (C18:3),
linoleoyl (C18:2), and oleoyl (C18:1)) acyl-CoAs. Cell extracts from
vG15Bac (hLPAAT
) baculovirus-infected insect cells, after 6 min of
reaction using 10 µM of acyl-CoAs, showed that the
highest activity was for palmitoleoyl-CoA (C16:1), which was 0.532 nmol
of CoA released per µg of protein (nmols/µg prot) (Fig.
5a), and then the rank order
was for the acyl-CoAs C16:0
C14:0
C12:0
C18:2 > C18:3
C18:0
C20:4 > C18:1 (0.389-0.285 nmol/µg of protein) with poor or no activity for the
acyl-CoAs C20:0 (0.180 nmol/µg of protein) and C24:0 (0.149 nmol/µg
of protein), respectively (Fig. 5a). The background activity of the wild type cell extracts, under these conditions, was from 0.135 (C20:0) to 0.190 (C18:2) nmol/µg of protein. When using 45 µM acyl-CoAs, the enzyme kinetics showed that hLPAAT
had maximal activity for the acyl-CoAs C16:0
C14:0
C16:1 > C18:2
C18:3
C12:0 (0.854-0.724
nmol/µg protein) (Fig. 5b) and intermediate activity for
the acyl chains C20:4, C18:1, and C18:0 (0.512, 0.496, and 0.452 nmol/µg of protein, respectively) (Fig. 5b). Again it showed poor or no activity for long acyl chains (C20:0 and C24:0) (0.262 and 0.148 nmol/µg of protein, respectively) (Fig.
5b). The background activity of the wild type cell extracts
when using 45 µM of acyl-CoAs was the same as that when
using 10 µM of acyl-CoAs (Fig. 5, a and
b). In these spectrophotometric assays, the recombinant hLPAAT
enzyme activity was linear for at least 3-6 min when using 45 µM acyl-CoAs or 1-2 min when using 10 µM acyl-CoAs (Fig.
6a and data not shown). The
wild type baculovirus background activity in these kinetic assays was
nearly constant irrespective of the time and amount or type of acyl-CoA
used (Fig. 6a and data not shown), suggesting that the wild
type baculovirus contains very little endogenous LPAAT activity.
Similar results of acyl donor specificity were obtained when the
reactions were performed using 3H-LPA, and after 6 min the
products were detected by TLC (Fig. 5c). When the TLC assay
was performed with unlabeled LPA and 14C-oleoyl-CoA,
specific labeled products (PA) were detected only when using vG15Bac
(hLPAAT
) cell extracts, which increased as the concentration of LPA
was increased (Fig. 6b).
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Cellular Localization--
To localize hLPAAT in the cell,
immunofluorescence on the different stable CHO cell lines was
performed. In permeabilized cells expressing the tagged hLPAAT
(CHOG15), an ER-staining pattern was observed using the T7.TagmAb (Fig.
8a) that was not present in
the control cell lines (CHO, CHOV) using the same mAb (data not shown).
A similar ER pattern was observed when the three cell lines were
stained with the specific ER mAb 1D3 that labels protein-disulfide isomerase from the ER (Fig. 8, b, c, and
d). The Predict-protein program predicts that, if hLPAAT
is also located in the plasma membrane, the T7.Tag should be in the
extracytoplasmic region, and thus, on nonpermeabilized cells, it would
be accessible and recognized by the specific T7.TagmAb. However,
immunostaining of unpermeabilized cells or fluorescence-activated cell
sorting analysis did not show external protein labeling, indicating
lack of plasma membrane localization (data not shown).
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DISCUSSION |
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We have cloned and sequenced the cDNA of the G15
gene, located in the MHC class III region, and have found that the
predicted polypeptide shares significant sequence identity with
bacterial, yeast, and plant LPAATs. We have also expressed the protein
product of that gene (hLPAAT) in insect cells and in the mammalian
cell line CHO and have shown that it contains LPAAT activity. We have seen that baculovirus recombinant hLPAAT
cannot incorporate
acyl-CoAs into LPC or LPE. However, using 11 different acyl-CoAs, we
have found that recombinant hLPAAT
converts LPA into different PAs depending on the acyl-CoA incorporated, and it shows different specificities for different acyl-CoAs. Our results indicate that the
hLPAAT
has affinity for fatty acids of acyl chain lengths from 12 to
18 carbons with a slight dependence on the degree of saturation of the
fatty acid. However, hLPAAT
does not incorporate long chain fatty
acids (C20:0, C24:0) unless they are saturated (C24:4). These hLPAAT
substrate specificity results confirm previous reported data by
Yamashita et al. (3, 5, 32) obtained using 10-20
µM acyl-CoAs, where the rank order of specificities of
the acyl donor was as follows: C18:1 > C18:2
C16:1 > C16:0
C14:0. However, C20:4, C18:0, and C12:0 were
described as poor substrates for LPAAT, although in our assays the
recombinant hLPAAT
has significant activity for these substrates. In
another study, Lands et al. (33), using rat liver microsomes
and 20 µM acyl-CoAs, described that the unsaturated
acyl-CoAs C18:3, C18:2, and C18:1 were the best substrates for LPAAT
followed by C16:0 and C18:0 and then, with low affinity, the
unsaturated 20-carbon fatty acids. The discrepancy between these
studies and our data may be due to the previous use of semipurified
(32) or unpurified (33) enzyme preparations from rat liver microsomes,
which could have been contaminated with other acyltransferases (3, 32),
such as hLPAAT
, whereas in our assay specificity is only due to the recombinant hLPAAT
.
Glycerophospholipids in animal tissues are known to contain large
amounts of arachidonic acid (C20:4) at the sn-2 position. It
has been proposed that PC would be deacylated at the sn-2
position and then reacylated, by the lyso-PC acyltransferase, with
C20:4 (3). Here, we have demonstrated that hLPAAT is able to
incorporate, with intermediate affinity, arachidonoyl-CoA (C20:4) into
LPA to form PA. The incorporation of C20:4 by hLPAAT
is of great relevance, since arachidonic acid, the most important prostaglandin precursor in humans, can be liberated from PA by the action of phospholipase A2. Prostaglandins mediate the
inflammatory response, the production of pain and fever, the regulation
of blood pressure, the induction of blood clotting, the control of
several reproductive functions, and the regulation of the sleep/wake
cycle (1).
Stamps et al. (17) have shown an increase in LPAAT activity
in COS7 cells transiently transfected with hLPAAT, indicating an
apparent preference of hLPAAT
for C18:1 monounsaturated CoA as acyl
donor. However, it is difficult to compare our data with theirs
because, although using the same activity assay, they used an excess of
substrates (10 times more LPA and 10 or 2.2 times more acyl-CoAs) in
their experiments. Comparison of our data on the acyl donor specificity
of hLPAAT
with that of Eberhardt et al. (18) on hLPAAT
could indicate that there is a difference in acyl donor specificity
between the two hLPAATs. hLPAAT
was found to show more activity in
the presence of the acyl donor C20:4, compared with the acyl donor
C16:0 (18), and our data indicate the reverse is true for hLPAAT
.
However, we have to be careful in interpreting these data, since
different LPAAT activity assays were used. Expression of the enzyme in
the baculovirus system has been very successful, since the wild type
baculovirus-infected cell extracts have shown very little endogenous
activity, and in consequence the LPAAT activity observed is only due to
the recombinant hLPAAT
. This expression system could be used in the future for the characterization of other acyltransferases, including hLPAAT
, to elucidate their particular specificities. We have used
homogenized cell extracts, instead of purified or detergent-treated enzyme, which maintain the hLPAAT
in its natural hydrophobic environment and allow a better biochemical and functional
characterization of the enzyme.
The initial steps in the biosynthesis of lipids have been described as
occurring in the cytosolic part of the ER (for a review, see Ref. 31).
We have shown that the hLPAAT is located in the ER using specific
mAbs that only recognize the T7.Tag fused to the recombinant enzyme.
This abolishes any possible cross-reaction with other endogenous
acyltransferases that could lead to misinterpretation of the results
when using Abs raised against the hLPAAT
polypeptide. We propose a
protein model in which hLPAAT
is an ER transmembrane protein where
the potential active center of the enzyme is facing the cytosolic part
of the ER. This model supports previous data obtained from microsomal
vesicles in which, by indirect biochemical methods (LPAAT inactivation
by proteases and the ability of LPAAT to bind to the
membrane-impermeable substrate palmitoyl-CoA), the active center of
LPAAT has been suggested to be located on the external (cytosolic) part
of the ER (31). Eberhardt et al. (18) propose that the
hLPAAT
contains four putative transmembrane helices with the first
and third oriented outside to inside and the second and fourth helices
oriented inside to outside, within the membrane. This orientation, if
we use it in relation to the ER membrane, is in agreement with the one
that we propose for hLPAAT
, since the protein region between the
third and fourth helices (containing our proposed active center that is
also conserved in hLPAAT
) would be in the cytosolic part of the
ER.
While a major function of phospholipids is to form biological
membranes, a subclass of phospholipids and their metabolites have been
implicated as signaling molecules, acting either as intracellular
second messengers or as extracellular agonists that modulate cell
function (34-37). LPAAT is the enzyme that converts LPA into PA. LPA
is an intracellular signaling molecule that is rapidly produced and
released by activated cells, notably platelets, to influence target
cells by acting on a specific cell surface receptor. LPA stimulates
platelet aggregation and cell proliferation and can be involved in
wound repair and blood clotting processes (34, 35). PA species can be
potent growth factor molecules; stimulate phospholipase C, protein, and
lipid kinases; mobilize Ca2+ flux; activate NADPH oxidase;
induce hormone release, platelet aggregation, and gene transcription
(37); and change cytoskeletal dynamics or cellular phenotype (see
references in Ref. 38). Recent studies have shown that IL-1 (39),
tumor necrosis factor-
(38), and platelet-activating factor (40), as
well as bacterial cell wall products such as lipid A or
lipopolysaccharide (41), may activate and signal, at least in part,
through a common lipid intracellular signaling pathway, leading to
rapid increases in intracellular levels of specific species of PA and
DAG by the activation of the enzyme LPAAT. Recently, LPAAT inhibitors
have been shown to block the inflammatory response produced by an
increase in PA (42). West et al. (16) have found that
overexpression of hLPAAT, in two different human cell lines, resulted
in an increase in LPAAT activity that correlated with enhancement of
transcription and synthesis of tumor necrosis factor-
and IL-6 from
cells upon stimulation with IL-1
. The finding of which hLPAAT is
involved in modulation of the inflammatory response will be crucial in relation to the designing of therapeutic agents.
Mobilization of intracellular stored Ca2+ by PA has been
proposed (for a review see Ref. 37), which in this case could be from
the ER, where hLPAAT is located, by the action of the induced PAs
produced by this enzyme. Bursten et al. (39) have suggested that, in addition to the ER, LPAAT should be located on the plasma membrane, since they observed a 1.3-fold activation of LPAAT by IL-1
when using plasma membrane-enriched compared with crude-microsomal cell
fractions. However, our data suggest that hLPAAT
is only located in
the ER but possibly very close to the plasma membrane (which could
contaminate membrane-enriched fractions). LPAAT could be activated by
direct contact with the plasma membrane or with receptors or receptor
complexes associated with the plasma membrane. A mechanism has been
described for the ryanodine receptor that, when located in the
sarcoplasmic reticulum, is activated by direct contact with the
dihydropyridine receptor located in the plasma membrane (43).
Human chromosomes 6 and 9 show gene family members present in both
chromosomes such as NOTCH-4, PBX-2, and
TN-X on chromosome 6p21.3 (class III region) and
NOTCH-1, PBX-3, and TN-C (tenascin-C) on 9q34 (44). We have found that the hLPAAT (G15) gene is
located on chromosome 6p21.3, while hLPAAT
is located on chromosome
9q34.3 (18), supporting the idea of duplication of an ancestral
chromosomal segment giving rise to the present chromosome 6p21 and
9q33-34 segments. A large number of diseases are associated with the
products of genes located in the MHC, many of which are not fully
explained by the class I and class II antigens. Further
characterization of hLPAAT
at the genetic and molecular levels could
extend the knowledge of the inflammatory response, lipid intracellular
signaling pathways, MHC, and disease association and result in the
rational design (based upon use of the recombinant protein) of novel
inhibitors that could suppress intracellular signals used by several
inflammatory mediators.
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ACKNOWLEDGEMENTS |
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We are most grateful to Antonio Alcamí for helpful discussions and critical reading of the manuscript and for help with the baculovirus expression system. We also thank John Broxholme for help with computing; Ana Pombo, Alain Vanderplasschem, and Michael Hollinshead for help with the confocal microscope; and Geoffrey Smith for access to specialized equipment.
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FOOTNOTES |
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* The confocal microscope (Sir William Dunn School of Pathology, Oxford) was funded by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y09565.
Supported by a European Community Training and Mobility Bursary
and a Florey-EPA Studentship.
§ To whom correspondence should be addressed: Tel.: 44-1865-275349; Fax: 44-1865-275729; E-mail: rdcampbell{at}molbiol.ox.ac.uk.
1 The abbreviations used are: LPAAT, lysophosphatidic acid acyltransferase; hLPAAT, human LPAAT; MHC, major histocompatibility complex; LPA, lysophosphatidic acid; PA, phosphatidic acid; ER, endoplasmic reticulum; DAG, diacylglycerol; PC, phosphatidylcholine; IL, interleukin; mAb, monoclonal antibody; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; bp, base pair(s); PCR, polymerase chain reaction; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid).
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REFERENCES |
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