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INTRODUCTION |
Phospholipase A2
(PLA2,1 EC
3.1.1.4) comprises a large family of structurally and mechanistically
distinct enzymes involved in membrane glycerophospholipid hydrolysis
(1). Phospholipases A2 that reside in the cytosol are of
particular interest because of their ability to interact directly with
signal transduction elements such as G-proteins and kinases. The
mammalian 85-kDa calcium-dependent cytosolic
PLA2 (cPLA2-
) is a well studied example of
this class of enzyme (2, 3). It is a monomeric polypeptide of 749 amino
acids containing an NH2-terminal
calcium-dependent regulatory domain responsible for the
translocation of the enzyme to membranes of the nuclear envelope upon
activation (4-6). The most convincing evidence for the involvement of
cPLA2-
in the release of arachidonic acid and the
initiation of subsequent proinflammatory response comes from mice that
have been made genetically deficient for cPLA2 (7, 8).
Stimulation of cells derived from these mice with calcium ionophore or
lipopolysaccharide results in little if any detectable increase in
leukotrienes, prostaglandins, or platelet-activating factor.
Importantly, these mice have a markedly reduced sensitivity to antigen
induced bronchoconstriction believed to be mediated at least in part by leukotrienes.
cPLA2-
lacks sequence similarity to other members
of the PLA2 super family. Although low-stringency
hybridization of genomic DNA failed to identify related genes, two
cPLA2- homologs, cPLA2-
and
cPLA2-
(9), have been identified in the EST data base using a cPLA2-
sequence as a query. In this report, we
describe the molecular cloning and initial biochemical characterization of cPLA2-
, which shares about 30% identity to
cPLA2-
.
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EXPERIMENTAL PROCEDURES |
Library Construction--
cDNA libraries were constructed
from U937 cells using a Poly(A) Track kit for isolation of mRNA
(Promega), a Superscript Choice kit for the generation of
double-stranded cDNA (Life Technologies, Inc.), and a LambdaZapII
phage cloning kit (Stratagene).
Molecular Cloning of Full-length cPLA2-
--
Two
cPLA2-
-specific deoxyribonucleotides were designed based
on the sequence of EST clone with GenBankTM accession
number W92213 (5'-CCTCCTGCAGCCCACTCGGGAC-3' and 5'-GCTGACCAGAGGAAAGTGCAGC-3').
These oligonucleotides were used to screen a U937 cDNA library. One
clone that hybridizes with both oligonucleotides, clone 52A, was
examined for complete DNA sequence determination. It contains a
4-kilobase pair insert with a partial coding sequence that begins at
nucleotide 1560 and continues to a stop codon at nucleotide 3894, representing 778 amino acids. The region on the DNA sequence 5' to
nucleotide 1560 was determined to be unspliced intron sequence. A
number of cDNAs isolated by hybridization have been examined in
this region and also shown to contain intron sequence.
A rapid amplification of cDNA ends (RACE) PCR strategy was used to
amplify the 5'-end sequence of cPLA2-
lacking the intron sequence. Double-stranded human heart cDNA with ligated adapters were purchased from CLONTECH. The cDNA was
amplified with primers NPCR1 (5'-GGCTGCAGGCCGTGGGCAGCCAGAGAGTCA-3') and
AP1 (the outer primer of the CLONTECH RACE PCR
kit), and the product was subsequently amplified with primers NPCR2
(5'-AGCAGTCAGAGGGGGTCACTAGGTCCTTAG-3') and AP2 (the inner primer from
CLONTECH). A major PCR band of about 700 base pairs
was purified on a 1% agarose gel and cloned into pCR-Blunt vector
(Invitrogen). The sequence for this fragment lacks the intron sequence
and extends the open reading frame by 234 additional amino acids. The
two gene segments were spliced together using a BfaI
restriction site in the region of overlap, creating a 3.4-kilobase pair
cDNA in pBluescript coding for 1012 amino acids
(pBlue-cPLA2-
wt).
Northern Blot Analysis--
Membranes blotted with human tissue
mRNA were purchased from CLONTECH and
32P-labeled probes derived from the coding region of clone
52A was used for hybridization. The washed membranes were exposed to
x-ray film to reveal the expression pattern of
cPLA2-
.
Production of Polyclonal Antibodies to
cPLA2-
--
Two regions of cPLA2-
,
representing amino acids 299-537 and 681-1012 were subcloned into
pGEX vectors for expression of glutathione S-transferase
fusion proteins in Escherichia coli (Amersham Pharmacia Biotech). Inclusion body material was prepared and used to raise polyclonal antibodies in rabbits.
Construction of Expression Vectors to Produce
cPLA2-
Proteins in COS-7 Cells--
The cDNA insert
of pBlue-cPLA2-
-wt was subcloned into mammalian
expression vector pED
C to generate plasmid
pED
C-cPLA2-
-wt. Transfection of this plasmid into COS
cells produced a full-length cPLA2-
protein of 110 kDa
as revealed by Western blot analysis of the harvested cell lysates. To
express cPLA2-
protein lacking the N-terminal 231 amino
acids, the 5' NcoI fragment of the cDNA insert in
pBlue-cPLA2-
-wt was deleted and the remaining cDNA insert was subcloned into pED
C to produce plasmid
pED
C-cPLA2-
-5'd2. Transfection of this plasmid into
COS cells resulted in expression of protein
cPLA2-
MEK, which was identified as a 90-kDa band
revealed by Western blot analysis.
Transfection cPLA2 Isoforms and Activity
Assay--
8 µg of pED
C vector, pED
C-cPLA2-
-wt,
pED
-cPLA2-
-5'd2, pEDdelta-cPLA2-
(9)
or pED-cPLA2-
plasmid DNA was transfected into COS-7
cells on a 10-cm cell culture plate using LipofectAMINE (Life
Technologies, Inc.) according to the manufacturer's protocol. At
66 h posttransfection, cells were washed twice with 10 ml of ice-cold Tris-buffered saline and scraped into 1 ml of Tris-buffered saline. Cell pellets were collected, resuspended in lysis buffer (10 mM HEPES, pH 7.5, 1 mM EDTA, 0.1 mM
dithiothreitol, 0.34 M sucrose, 1 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin), and lysed in a
Parr-bomb (700 psi, 10 min) on ice. Protein concentration was measured
by Bradford assay (Bio-Rad), and lysis buffer was used to adjust the
samples to the same protein concentration. Phospholipase assays were
carried out as described (9).
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RESULTS AND DISCUSSION |
Identification of cPLA2-
and cDNA
Cloning--
As described previously, search of the
GenBankTM EST data base using the human 85-kDa
cPLA2- protein sequence (cPLA2-
) as a query
yielded two ESTs with related sequences, which we named cPLA2-
(GenBankTM accession number W92213)
and cPLA2-
(GenBankTM accession number
N56796). Through combined efforts of cDNA library screening and
RACE PCR amplification, we obtained a cPLA2-
cDNA
clone containing a complete open reading frame encoding a polypeptide
of 1012 amino acids with a calculated molecular mass of 110 kDa. A
comparison of the amino acid sequence of cPLA2-
with
that of cPLA2-
(2) and cPLA2-
(9)
revealed the following features. 1) All three enzymes share a
homologous catalytic region which can be divided into domain A and
domain B (Fig. 1A). Domain A
contains 253, 251, and 237 amino acids for cPLA2-
,
cPLA2-
, and cPLA2-
, respectively. The
lipase consensus sequence, GXSXG, is located at
the N-terminal region of domain A (indicated in Fig. 1A). A
signature of
GGGXRAX5GX6EXGLLDX6GXSGSTWX4LY
around the lipase consensus sequence can be assigned for the three
cPLA2- isoforms (Fig. 1B). Catalytic domain B
contains 196, 186, and 170 amino acids for cPLA2-
,
cPLA2-
, and cPLA2-
, respectively. The
linker regions between domains A and B are from 91 amino acids (for
cPLA2-
) to 133 amino acids (for cPLA2-
)
with very limited homology (Fig. 1B). It is interesting to
note that the three residues in the putative catalytic triad of
cPLA2-
(10) are all found in the conserved domains A
(arginine and serine) and B (aspartate), whereas the phosphorylated
serines that are believed to participate in cPLA2-
regulation (11, 12) are all located in the less conserved interdomain
region and the COOH-terminal tail (Fig. 1, A and
B). 2) Both cPLA2-
and cPLA2-
have a calcium-dependent lipid binding (CaLB) domain that
is lacking in cPLA2-
. However, cPLA2-
has
a 120 amino acid insert between the CaLB domain and catalytic
domain A, whereas in cPLA2-
the two domains are adjacent to each other. In addition, cPLA2-
has an
NH2-terminal domain composed of 242 amino acids, which
shares no homology to any protein sequence in the GenBankTM
data bases.


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Fig. 1.
Sequence comparison of three
cPLA2 isoforms. A, schematic diagram of
homologous protein domains. The number of amino acids for each domain
are indicated in the boxes. The percentage of identical
amino acids to cPLA2- is shown above the homologous
domains in cPLA2- and cPLA2- . The
stars denote the conserved serines in the lipase consensus
sequence shown above the catalytic domain A of each isoform. The
phosphorylated serines of cPLA2- and their positions are
also shown. B, alignment of amino acid sequences of the
three cPLA2-s. The conserved domains are marked by
two arrows, and the three residues in the putative catalytic
triad are denoted by stars. Identical amino acids are
shaded. The sequence of cPLA2- has been
submitted to GenBankTM under accession number
AF121908.
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When the multiple human tissue mRNA blots were hybridized
with a probe derived from the cPLA2-
cDNA containing
sequences from the CaLB domain to the COOH terminus (see
"Experimental Procedures"), a discrete band of about 8 kilobase
pairs can be observed in all the tissues examined (Fig.
2). This indicates that
cPLA2-
mRNA is expressed ubiquitously, although at
higher levels in pancreas, brain, heart, and liver tissues.

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Fig. 2.
Expression of
cPLA2- mRNA in different human
tissues by Northern analysis. Human multiple-tissue blots
(CLONTECH) were hybridized with
32P-labeled cPLA2- -specific probes and
washed under high stringency conditions. The same blots were also
probed with -actin DNA and confirmed to have equal amounts of
mRNA loaded in each lane (data not shown).
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Expression of cPLA2-
in COS Cells and
PLA2 Activity Assay--
When full-length
cPLA2-
cDNA was subcloned into a eukaryotic
expression vector and used to transfect COS-7 cells, a
cPLA2-
-specific protein of about 110 kDa was detected in
cell lysates as revealed by Western analysis using polyclonal
antibodies raised against bacteria-expressed cPLA2-
peptide (data not shown). When the cell lysates were tested in a
PLA2 activity assay using
1-palmitoyl-2-[14C]arachidonyl-PC (P*APC) as a substrate
(see "Experimental Procedures"), the amount of arachidonic acid
released by cPLA2-
-transfected lysate is only twice as
much as that by vector-transfected cell lysate (Fig.
3). We suspected that the relatively
small increase of PLA2 activity might be due to the
relative low level expression of a large protein, or the N-terminal
domain of cPLA2-
could be inhibiting the enzyme activity
of the catalytic domains. Therefore, we constructed the
cPLA2-
MEK plasmid expressing cPLA2-
protein lacking the N-terminal 236 amino acids (starting from
Met-Glu-Lys sequence just upstream of the CaLB domain). Indeed, COS
cell lysate transfected with cPLA2-
MEK has an
approximate 6-fold increase in PLA2 activity compared with
vector-transfected cell lysate. However, Western analysis of cell
lysates using cPLA2-
antibodies followed by scanning
quantification reveals that 5-fold more cPLA2-
MEK protein was expressed compared with the full-length
cPLA2-
(data not shown). These results suggest that
cPLA2-
proteins do have PLA2 activity, and
cPLA2-
MEK has the same specific activity as cPLA-
protein, indicating that the N-terminal domain is not negatively regulating the activity of the catalytic domains. When lysates from COS
cells transfected with either cPLA2-
or
cPLA2-
MEK were subjected to ultracentrifugation
(100,000 × g centrifugal force), the majority
(60-70%) of the cPLA2-
protein and phospholipase activity was found in the supernatant fraction as detected by Western
blot analysis and P*APC hydrolysis assay (data not shown), suggesting
that cPLA2-
is a cytosolic protein. In addition, when COS cell lysate transfected with a CaLB-containing
cPLA2-
construct was fractionated in the presence of
calcium, nearly all the enzyme activity was found to be associated with
the particulate fraction (data not shown). Therefore we predict
cPLA2-
may respond to a calcium-mobilizing agonist in
similar fashion as cPLA2-
.

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Fig. 3.
Overexpression of cPLA2 isoforms
in COS cells correlates with increased PLA2 activity.
Lysates from COS cells transfected with either cPLA2-
( ), cPLA2- MEK ( ), or vector DNA ( ) were assayed
for their ability to release arachidonic acid from P*APC at 37 °C
for indicated times. Activity is expressed as percentage of radiolabel
released (% conversion).
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Relative Regiospecificity of cPLA2-
--
Because of
a higher level of expression, cPLA-
MEK rather than the full-length
cPLA2-
protein was used to compare regioselectivity of
the three cPLA2- isoforms. For this purpose, one of the
three substrates, namely
1-[14C]palmitoyl-2-arachidonyl-PC (*PAPC),
1-palmitoyl-2-[14C]arachidonyl-PC (P*APC), and
1-O-hexadecyl-2-[3H]arachidonyl-PC (O-H*APC), was used in
a vesicle assay system to measure radiolabeled fatty acids released by
COS cell lysates transfected with either cPLA2-
,
cPLA2-
MEK, cPLA2-
, or vector plasmid DNA.
Although all three enzymes were able to liberate the sn-2
arachidonic acid from P*APC, each enzyme has unique properties in
releasing the radioactive labels in *PAPC and O-H*APC.
cPLA2-
MEK and cPLA2-
were able to
liberate the sn-1 palmitic acid from *PAPC, although
cPLA2-
had almost no activity in this regard (Fig.
4). On the other hand,
cPLA2-
and cPLA2-
could release the
sn-2 arachidonate from O-H*APC, but cPLA2-
MEK
showed very limited activity. Although full-length
cPLA2-
is expressed at low levels, preferences toward
the three substrates are the same as cPLA2-
MEK (data not
shown). These results indicate that under conditions tested,
cPLA2-
prefers sn-2 cleavage,
cPLA2-
prefers sn-1 cleavage, whereas
cPLA2-
is able to efficiently cleave at either site.
This implies that the observed PLA2 activity of
cPLA2-
(Fig. 3) is likely due to combined
PLA1 activity and lysophospholipase activity (or
transacylase activity). However, affirmative conclusion can only be
made when extensive enzymology studies are conducted with pure
cPLA2-
proteins.

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Fig. 4.
Phospholipase A1/A2
activities of cPLA2 isoforms. Lysates from COS cells
transfected with either cPLA2- (A),
cPLA2- MEK (B), cPLA2-
(C), or vector DNA were incubated with *PAPC ( ), P*APC
( ), and O-H*APC ( ) for the indicated time periods. Activity is
expressed as percentage of conversion.
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Phospholipase Activity of cPLA2-
Is Calcium
dependent--
Because cPLA2-
has a CaLB
domain, we tested whether its phospholipase activity is
calcium-dependent. As can be seen from Fig.
5, when *PAPC (Fig. 5A) or
P*APC (Fig. 5B) was used as substrate, release of the
radiolabeled fatty acids by both cPLA2-
and
cPLA2-
MEK is strictly dependent on the presence of
calcium. When EGTA was included in the reaction, no phospholipase
activity was detected. This experiment demonstrates that the CaLB
domain in cPLA2-
is functional. We were unable to
determine the minimal calcium concentration required for enzymatic
activation due to impurities of the cell lysates.

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Fig. 5.
Phospholipase activity of
cPLA2- is
calcium-dependent. COS lysates from cells transfected
with cPLA2- , cPLA2- MEK, or vector were
assayed for their ability to liberate radioactive labels from *PAPC
(A) or P*APC (B) in the presence of 7 mM calcium (shaded bars) or 5 mM
EGTA (open bars). Activity is measured as percentage of
radiolabel released (% conversion).
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In conclusion, we have cloned a cDNA for a 110-kDa
calcium-dependent phospholipase, cPLA2-
,
which shares significant homology to cPLA2-
and
cPLA2-
. Although the three members of cPLA2
family share similar catalytic domains and have the same conserved
residues in the catalytic triad (10), initial biochemical
characterization suggests that they have different regioselectivity and
may also have different headgroup specificity. The mechanisms for
membrane association are also different: cPLA2-
, like
cPLA2-
, seems to rely on the CaLB domain to gain access
to aggregated phospholipids, whereas cPLA2-
may use
lipid modification for membrane attachment. Furthermore, the fact that
cPLA2-
has additional protein domains suggests that the
regulation of this enzyme may be dramatically different from that of
cPLA2-
and cPLA2-
.