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INTRODUCTION |
Phosphatidylserine (PS)1
in cell membranes is known to be an essential cofactor for the
activation of protein kinase C (1) and for blood coagulation (2). More
recently, PS has been shown to regulate the activity of various
enzymes, such as c-Raf-1 protein kinase (3), nitric oxide synthase (4),
Na+/K+-ATPase (5), dynamin GTPase (6), and
diacylglycerol kinase (7). PS is predominantly located on the inner
leaflet of plasma membranes in various types of cells (8) but appears
on the outer leaflet after stimulation by various factors such as
cytokines (9, 10), inflammatory reactions, and platelet activation (8,
11-13). Surface-exposed PS has also been shown to act as a signal for
the removal of damaged or aged cells by the reticuloendothelial system
and is observed in cells undergoing apoptosis (14, 15). Thus, the
exposure of PS on the cell surface must be tightly regulated.
Another serine-containing phospholipid, lyso-PS, is implicated to act
as a lipid mediator under pathophysiological conditions (16). For
example, lyso-PS is demonstrated to interact with local mast cells
(17), producing specific and stereoselective activation (18). It also
induces transient increases in cytosolic free Ca2+
([Ca2+]i) in ovarian and breast cancer cells (19)
and lyso-PS; 2-acyl-1-lyso-PS with unsaturated fatty acids especially
inhibits mitogen-induced T cell activation (20). Lyso-PS is present in human serum, the aqueous humor and the lachrymal gland fluid of the eye
(21). It is likely to be produced from PS by phospholipase A1 or A2, but the precise mechanisms of lyso-PS
production and elimination in vivo remain to be clarified.
We previously demonstrated that a serine phospholipid-specific
phospholipase A1/lysophospholipase is secreted from rat
platelets when they are activated (22, 23). Very recently we purified this enzyme from rat platelets and cloned its cDNA (24). Although this novel phospholipase, named PS-PLA1, has a similar
structure to members of the lipase family such as hepatic, pancreatic,
and lipoprotein lipases, PS-PLA1 does not hydrolyze
triacylglycerol but specifically acts on PS or 1-acyl-2-lyso-PS to
hydrolyze fatty acids at the sn-1 position of these
phospholipids (24). Thus, PS-PLA1 mediates two types of
reactions, producing 2-acyl-1-lyso-PS from PS and eliminating
1-acyl-2-lyso-PS. It is secreted from cells and has an affinity for
heparin, like other members of the lipase family (24). It is likely
that PS-PLA1 is involved in regulating
PS/lyso-PS-dependent reactions under physiological conditions. Indeed, PS-PLA1 can hydrolyze PS in rat
platelets when the cell is activated (23). The physiological function of PS-PLA1, however, is still unknown and requires further
investigation. As a part of our continuing study of the physiological
role of PS-PLA1, we have isolated the cDNA for human
PS-PLA1. In the course of the study, we detected an
alternative splicing form of human PS-PLA1 and identified
it as a lyso-PS-specific lysophospholipase.
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MATERIALS AND METHODS |
cDNA Cloning of Human PS-PLA1--
With the
polymerase chain reaction (PCR), we amplified cDNA using a human
liver
gt11 cDNA library (CLONTECH, Palo
Alto, CA) as a template, and the following primers, based on the EST
clone: 5'-CACGAGGATTTCCAGCTCAG-3' and 5'-CCCTGTACATGTTTCTATTG-3'.
The resulting 1.7-kb PCR fragment was used as a DNA probe to screen the
human liver
gt11 cDNA library as described previously (24). One
positive clone was identified, and its insert cDNA was subcloned
into the pBluescript II SK phagemid vector (Stratagene, La Jolla, CA).
The DNA sequence was determined by the dideoxynucleotide chain
termination method using a Dye Terminator Cycle Sequencing FS Ready
Reaction Kit (Perkin-Elmer) and an ABI PRISM 377 DNA sequencer
(Perkin-Elmer).
Southern Blot Analysis--
Human and rat genomic DNAs (12 µg
each) were digested with the appropriate restriction enzymes (50 units
each) for 12 h then run on a 0.8% SeaKem Gold Agarose gel (FMC
BioProducts, Rockland, ME). The DNA was then transferred under
capillary pressure to a Hybond-N nylon hybridization transfer membrane
(Amersham Pharmacia Biotech). The relevant DNA probes were labeled by
random priming with [
-32P]dCTP. Hybridization was
carried out at 65 °C for 4 h in a rapid-hybridization buffer
(Amersham Pharmacia Biotech). The blot was rinsed in 2× SSC (1×
SSC = 0.15 M NaCl and 0.015 M sodium
citrate) at room temperature for 5 min, then washed twice in 0.5× SSC,
0.1% SDS at 65 °C for 40 min. The blot was exposed to Kodak X-Omat
AR film at
80 °C with an intensifying screen for 12 h.
Northern Blot Analysis--
Human multiple tissue Northern blots
were purchased from CLONTECH. Total RNA from human
platelets (10 µg/lane) was separated by 1%
agarose-formaldehyde gel electrophoresis and transferred onto
Hybond-N. Hybridization with the human PS-PLA1 probe and washing were carried out as described for the Southern blot analysis. The blots were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe (CLONTECH) as an
internal standard.
Amplification and Restriction Enzyme Digestion of Human
PS-PLA1 cDNA--
Total RNA (1 µg) obtained from
various human tissues and cell lines was used as a template in a
reverse transcriptase (RT)-PCR. A Ready-To-Go T-Primed First-Strand Kit
(Amersham Pharmacia Biotech) was used according to the manufacturer's
protocol to synthesize the first-strand cDNA. DNA fragments were
amplified by PCR using 3 µl of the first-strand reaction product as a
template and the following set of primers:
5'-ACAAGGACACCAACATCGAGGTTACCTTCC-3' (nucleotide positions 1055-1084
of the cDNA encoding human PS-PLA1) and
5'-CAGTCACACTTGCTTGTAAGTTCACTGG-3' (nucleotide positions 1312-1339). The RT-PCR products (285 or 289 bp) were digested with BsrGI
for 4 h at 60 °C then subjected to 4% NuSieve 3:1 Agarose (FMC
BioProducts) gel electrophoresis.
Expression of Human PS-PLA1 in Sf9
Cells--
DNA fragments encoding the normal and truncated forms of
human PS-PLA1 were amplified by PCR using two synthetic
oligonucleotides. The 5' oligonucleotide,
5'-CAGCGGATCCATGCCCCCAGGTCCCTGGGA-3', contained the
BamHI site in addition to the human PS-PLA1
sequence, whereas the 3' oligonucleotide,
5'-AAAAAAGCTTGCAGGGAGATGTGTCCTGCCCAGG-3', contained
the HindIII site and a complementary human
PS-PLA1 sequence. The amplified cDNA fragments were
subcloned into the BamHI and HindIII sites of the
pFASTBAC1 expression vector (Life Technologies, Inc.) to generate donor
plasmids. Recombinant viruses were then prepared using the BAC-TO-BAC
baculovirus expression system (Life Technologies, Inc.) according to
the manufacturer's protocol. The resulting recombinant baculovirus was
used to infect Sf9 cells. Four days after infection, the culture
supernatant of the infected cells was collected, and
PS-PLA1 activity was determined as described below.
Lysophospholipase and Phospholipase A1
Assay--
Lysophospholipase activity of PS-PLA1 and
PS-PLA1
C was measured as described previously (24).
Briefly, 1-oleoyl-2-lyso-PS, 1-oleoyl-2-lysophosphatidylcholine,
1-oleoyl-2-lysophosphatidic acid, and
1-oleoyl-2-lysophosphatidylethanolamine (40 µM each) containing a 14C-labeled fatty acid at the sn-1
position were incubated at 37 °C for 15 min with aliquots of various
fractions of heparin fast protein column chromatography in 100 mM Tris-HCl (pH 7.5) with 0.4 mM
CaCl2. Dioleoyl-PS containing a 14C-labeled
fatty acid at the sn-1 position was prepared as described previously (23). The PS (40 µM) was incubated at 37 °C
for 15 min with aliquots of various fractions of heparin fast protein column chromatography in 100 mM Tris-HCl (pH 7.5) with 4 mM CaCl2. The fatty acid liberated was
extracted by the modified Dole's method, and radioactivity was
measured by scintillation counter.
Column Chromatography--
The culture supernatant of Sf9
cells or human plasma was loaded onto a HiTrap Heparin (1 ml) affinity
column (Amersham Pharmacia Biotech) that had been preequilibrated with
100 mM NaCl containing 10 mM Tris-HCl (pH 7.4)
using the fast protein liquid chromatography system (Amersham Pharmacia
Biotech). The column was washed thoroughly with this buffer and eluted
with a linear gradient of NaCl (0.1-1.1 M). The
PS-PLA1 activity in each fraction was determined as
described above.
Analysis of the Gene Structure of Human
PS-PLA1--
PCR was performed using human genomic DNA as
a template and the primers 5'-GAGAAACAAGGACACCAACATCGAGGTTACC-3' and
5'-CATTGTAGGGTGGCATGGGCTATGATTCC-3', based on the sequence around the
insertion. The DNA sequence of the resulting 3.5-kb fragment was
determined by direct sequencing using the above oligonucleotides as
sequence primers.
Chromosomal Preparation and in Situ Hybridization--
We used
the direct R-banding fluorescence in situ hybridization
method to determine the human chromosomal location of the PS-PLA1 gene. We prepared R-banded chromosomes
and performed fluorescence in situ hybridization as
described previously (25). A mitogen-stimulated lymphocyte culture
was synchronized by thymidine blockade, with 5-bromodeoxyuridine
incorporated during the late replication stage to allow differential
replication staining after removing excessive thymidine. R-band
staining was performed by exposing chromosome slides to UV light after
staining with Hoechst 33258.
The chromosome slides were hardened at 65 °C for 2 h, then
denatured at 70 °C in 70% formamide in 2 × SSC and dehydrated
in a 70, 85, 100% ethanol series at 4 °C. The 1.75-kb human
cDNA fragment inserted into pBluescript II SK was used as a probe. The cDNA fragments were labeled by nick translation with biotin 16-dUTP (Boehringer Mannheim) following the manufacturer's protocol. The labeled DNA fragment was ethanol-precipitated with salmon sperm DNA
and Escherichia coli tRNA, then denatured at 75 °C for 10 min in 100% formamide. The denatured probe was mixed with an equal
volume of hybridization solution to produce a final concentration of
50% formamide, 2× SSC, 10% dextran sulfate, and 2 mg/ml bovine serum
albumin (Sigma). Twenty µl of this mixture containing 250 ng of
labeled DNA was placed onto the denatured slide, covered with Parafilm,
and incubated overnight at 37 °C. The slides were washed in 50%
formamide in 2× SSC at 37 °C, then in 2× SSC, and finally in 1×
SSC (for 20 min each time) at room temperature. After rinsing in 4×
SSC, they were incubated under a coverslip with goat anti-biotin
antibody (Vector Laboratories) at a dilution of 1:500 in 1% bovine
serum albumin, 4× SSC, then with 4× SSC (for 5 min each time). The
slides were then stained with fluorescein-anti-goat IgG (Nordic
Immunology) at a dilution of 1:500 for 1 h at 37 °C. After
washing with 4× SSC, then 0.1% Nonidet P-40 in 4× SSC, and again
with 4× SSC (for 10 min each time) on a shaker, the slides were rinsed
with 2× SSC and stained with 0.75 µg/ml propidium iodide. Excitation
at a wavelength of 450-490 nm (Nikon filter set B-2A) and near 365 nm
(UV-2A) was then measured. Kodak Ektachrome ASA100 films were used for microphotography.
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RESULTS |
Molecular Cloning of Human PS-PLA1--
Southern blot
analysis showed the existence of a single gene copy in humans and rats
(data not shown). We used the BLASTN program to search the protein data
base for sequences similar to that of rat PS-PLA1 and found
two human EST sequences with homology to rat PS-PLA1 on the
GenBankTM DNA data base (GenBankTM accession numbers T96213 (5' region)
and T96131 (3' region)). We prepared one set of PCR primers
corresponding to the 5'-untranslated region and the 3'-untranslated
region based on the DNA sequences of these EST clones. Because the EST
clones were derived from a human fetal liver and spleen cDNA
library, we performed PCR using human liver cDNA as a template. We
then used the resulting 1.7-kb DNA fragment to screen a human liver
cDNA library, and one positive clone was isolated. DNA sequence
analysis revealed that this clone has a sequence highly homologous with
that of rat PS-PLA1, covering the whole region
corresponding to the open reading frame (Fig.
1a). This cDNA clone
contained a 1368-bp open reading frame that encoded 456 amino acids,
starting with an initiation codon (ATG) at nucleotide 32 (numbered as
1) and ending with a stop codon (TAG) at position 1369-1371. This open
reading frame was flanked by 5'- and 3'-untranslated sequences of 31 and 359 bp, respectively, with a polyadenylation signal 19 bp before
the poly(A)+ tail. Comparison of this open reading frame
with rat PS-PLA1 revealed extensive homology at both the
nucleotide (82.1%) and amino acid (80.0%) levels. Ser-142, Asp-166,
and His-236, which may make up a catalytic triad in rat
PS-PLA1 (24), are conserved between the rat and human
enzymes. The amino acid sequences around these three amino acids were
also highly conserved between the two species. A putative lid, which is
composed of 22 or 23 amino acid residues in most of the other lipases
and suggested to be involved in both substrate recognition and surface
activation, is composed of 12 residues, as for the rat enzyme.

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Fig. 1.
a, nucleotide and amino acid sequences
of human PS-PLA1. The first and
second lines indicate the nucleotide and the
deduced amino acid sequence, respectively. The nucleotide and amino
acid positions are shown at both sides. The putative lid residues are
underlined, and the polyadenylation signal is double
underlined. b, nucleotide and amino acid sequences of
human PS-PLA1 C. The sequences of PS-PLA1 C
that differ from those of PS-PLA1 are shown. The four extra
bases, GTAC, are shaded. c, schematic model of
PS-PLA1 and PS-PLA1 C. The positions of the
active serine, aspartic acid, and histidine residues that compose the
catalytic triad of PS-PLA1 are shown by hatched
bars.
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When the coding region of PS-PLA1 mRNA from various
human tissues and cell lines was amplified using RT-PCR and analyzed by DNA sequencing, an aberrant mRNA was frequently detected in
addition to PS-PLA1 mRNA. These clones, designated
PS-PLA1
C, had four additional bases (GTAC) inserted at
nucleotide position 1122 of PS-PLA1, and the other
nucleotide sequence of this clone was identical to that of
PS-PLA1. As the insertion of these four bases caused a
frameshift (Fig. 1b), PS-PLA1
C loses about 80 amino acid residues that are present in C terminus of
PS-PLA1, but the amino acids forming a catalytic triad
including Ser-142, Asp-166, and His-236 are all preserved, suggesting
that PS-PLA1
C is catalytically active (Fig.
1c).
Production of Two Isoforms by Alternative Splicing--
We
analyzed the structure of the gene and nucleotide sequences around the
insertion point, as described under "Materials and Methods." The
four extra bases, GTAC, inserted in the PS-PLA1
C cDNA were found at the beginning of the intron (Fig.
2a). Two separate consensus
sequences for RNA splicing (gt(a/g)agt) (Fig. 2b) can be
found at this exon-intron boundary. It is likely from this information
that mRNA for PS-PLA1 is produced when the first consensus site (gtacgt) is used, and mRNA for
PS-PLA1
C is produced when the second (gtaagt) is used
(Fig. 2c). Thus, we concluded that PS-PLA1 and
PS-PLA1
C arise from alternative use of the 5'-splicing donor sites (GT).

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Fig. 2.
Alternative splicing of
PS-PLA1. a, the nucleotide sequence of the
human and rat PS-PLA1 genes around the insertion
point of the four extra bases. The capital letters represent
the exon, and the small letters represent the intron for
PS-PLA1. The four bases,
gatc (in italics), were found at the 5'-end of
the exon-intron boundary. The nucleotide numbers in Fig. 1 were shown
for PS-PLA1 and PS-PLA1 C, respectively.
b, possible exon-intron structure of the human
PS-PLA1 gene. The 5'-splicing donor sites are
underlined. Consensus sequences for RNA splicing are shown
in the lower panel. The nucleotide numbers in Fig. 1 were
shown for PS-PLA1 and PS-PLA1 C,
respectively. c, model for alternative splicing of
PS-PLA1. PS-PLA1 mRNA is produced when the
first 5'-splicing donor site (gtacgt) is used; mRNA for the
PS-PLA1 C is produced when the second site (gtaagt) is
used. The nucleotide numbers in Fig. 1 were shown for
PS-PLA1 and PS-PLA1 C, respectively.
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PS-PLA1
C as a Lyso-PS-specific
Lysophospholipase--
To determine whether the products encoded by
the two mRNAs have PS- or lyso-PS-hydrolyzing activity, recombinant
PS-PLA1 and PS-PLA1
C were produced using a
baculovirus system and examined for activities. As shown in Fig.
3a, we detected appreciable
lyso-PS-hydrolyzing activity in the supernatant of Sf9 cells
infected with either PS-PLA1 or PS-PLA1
C
recombinant viruses. This shows that both PS-PLA1 and
PS-PLA1
C are secreted from these cells and possess enzymatic activity to hydrolyze lyso-PS. By contrast, no PS-hydrolyzing activity was observed in the supernatant of cells infected with the
PS-PLA1
C recombinant virus (Fig. 3a). We also
examined the substrate specificity using lyso-PS,
1-oleoyl-2-lysophosphatidylcholine, 1-oleoyl-2-lysophosphatidylethanolamine, and
1-oleoyl-2-lysophosphatidic acid. As shown in Fig. 3b,
both PS-PLA1 and PS-PLA1
C hydrolyzed lyso-PS, but neither produced appreciable hydrolysis of other lysophospholipids. Thus, PS-PLA1 hydrolyzes both PS and
lyso-PS, but PS-PLA1
C exhibits lyso-PS-specific
lysophospholipase activity.

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Fig. 3.
Biochemical characterization of
PS-PLA1 C. a,
PS-PLA1 C does not hydrolyze PS. PS-PLA1 and
PS-PLA1 C recombinant proteins were partially purified
from the culture supernatants of Sf9 cells infected with either
PS-PLA1 and PS-PLA1 C recombinant baculovirus
and were used as an enzyme source. The PS- or lyso-PS-hydrolyzing
activities of PS-PLA1 are indicated as 100%. b,
PS-PLA1 C is a lyso-PS-specific lysophospholipase. The
substrate specificity of PS-PLA1 and
PS-PLA1 C was examined using lyso-PS,
1-oleoyl-2-lysophosphatidic acid,
1-oleoyl-2-lysophosphatidylethanolamine, and
1-oleoyl-2-lysophosphatidylcholine as substrates and partially purified
PS-PLA1 and PS-PLA1 C as enzyme sources.
Shaded bar, lyso-PS; open bar,
1-oleoyl-2-lysophosphatidic acid; hatched bar,
1-oleoyl-2-lysophosphatidylethanolamine; filled bar,
1-oleoyl-2-lysophosphatidylcholine. c, affinity of
PS-PLA1 and PS-PLA1 C for heparin. The
culture supernatants of Sf9 cells infected with
PS-PLA1 or PS-PLA1 C recombinant baculovirus were applied onto a HiTrap heparin fast protein
liquid chromatography column, and PS-PLA1 and
PS-PLA1 C were eluted using a linear gradient of NaCl.
The lysophospholipase activity of each fraction was measured using
lyso-PS as a substrate.
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We have previously demonstrated that rat PS-PLA1 possesses
a heparin binding site, because this enzyme absorbed to a
heparin-Sepharose column (24). We found that both human
PS-PLA1 and PS-PLA1
C were also absorbed to
the column and eluted with increasing concentrations of NaCl. The
elution profile of PS-PLA1
C on heparin-Sepharose column
chromatography was almost identical to that of PS-PLA1 (Fig. 3c), indicating that the heparin binding site of
PS-PLA1 is located in N-terminal region of this enzyme.
Expression of PS-PLA1 and PS-PLA1
C in
Human Tissues and Cells--
First we used Northern blot analysis to
examine the expression of PS-PLA1 mRNA
(PS-PLA1 and PS-PLA1
C) using the cDNA
corresponding to the coding region as a probe (Fig.
4). Transcripts of 1.9-kb messages were
seen in most of the tissues examined, with the highest expression in
the liver and prostate gland. PS-PLA1 mRNAs were not
detected in leukocytes or platelets (Fig. 4). Because
PS-PLA1 was first identified in rat platelets, we analyzed
the expression of PS-PLA1 in human platelets by RT-PCR
analysis and an assay for PS-PLA1 activity. No appreciable
PS-PLA1 activity or transcripts were detected in human
platelets (data not shown).

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Fig. 4.
Northern blot analysis of PS-PLA1
in human tissues and blood cells. Poly(A)+ RNA (2 µg) from various human tissues (human multiple tissue Northern blots,
CLONTECH) and 10 µg of total RNA from human
platelets were hybridized with probes specific to human
PS-PLA1 (upper panel) and
glyceraldehyde-3-phosphate dehydrogenase (lower panel) on a
nylon membrane. The origins of each RNA are shown at the top. Molecular
mass standard markers are shown on the left.
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The PS-PLA1 transcript present in human tissues was
detected as a single band on Northern blot analysis (Fig. 4). To
determine the relative amounts of PS-PLA1 and
PS-PLA1
C mRNAs in human tissues, RT-PCR analysis was
performed using total RNAs derived from various human tissues. Because
the insertion of the four extra bases generates a new restriction
enzyme site, BsrGI (TGTACA) (Figs. 1 and 2), we quantified
the frequency of the two mRNAs by digesting the RT-PCR products
with BsrGI, as described under "Materials and Methods."
As shown in Fig. 5, both
PS-PLA1 and PS-PLA1
C transcripts were
detected in various human tissues, including skeletal muscle, kidney,
small intestine, spleen, and testis. The amount of
PS-PLA1
C in these tissues was about 10 to 20% that of
the PS-PLA1 level (Fig. 5). We also examined the expression
of PS-PLA1 and PS-PLA1
C in several human
cell lines. mRNAs for both PS-PLA1 and
PS-PLA1
C were detected in human fibroblast,
keratinocyte, melanoma, HepG2, and HeLa cells. Thus, human tissues and
cell lines express both PS-PLA1 and
PS-PLA1
C, although the relative amount of
PS-PLA1
C is lower than that of PS-PLA1.

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Fig. 5.
Expression of the two alternatively spliced
transcripts of PS-PLA1 in human tissues and cells
determined by RT-PCR. mRNAs for both PS-PLA1 and
PS-PLA1 C were detected by RT-PCR. The PCR products were
subjected to BsrGI digestion, as the BsrGI site
only occurs in PS-PLA1 C. In the control experiment,
plasmids carrying PS-PLA1 or PS-PLA1 C were
used as a template. After BsrGI digestion, the
PS-PLA1 plasmid gave rise to a single 285 bp band, whereas
the PS-PLA1 C plasmid gave rise to two bands of 220 and
69 bp. Bands of 220 and 69 bp (derived from PS-PLA1 C)
were detected in addition to a band of 285 bp (derived from
PS-PLA1) in various tissues and cell lines.
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Chromosome Mapping of PS-PLA1--
The location of the
PS-PLA1 gene on the human chromosome was
assigned by direct R-banding fluorescence in situ
hybridization using a human cDNA fragment as a probe. The
PS-PLA1 gene was localized to human chromosome
3q13.13-13.2 (Fig. 6).

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Fig. 6.
Chromosomal localization of human
PS-PLA1 gene. Localization of the
PS-PLA1 gene on human R-banded chromosomes was
determined using a 1.75-kb human cDNA fragment as a biotinylated
probe as described under "Materials and Methods." The hybridization
signals are indicated by arrows. The signals are localized
to human chromosome 3q13.13-13.2. The metaphase spreads were
photographed with a Nikon B-2A filter. Two positive signals in separate
fields were shown (a and b).
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DISCUSSION |
PS-PLA1
C as a Lyso-PS-specific
Lysophospholipase--
So far it has been reported that the various
phospholipases for which the cDNA structure is already known,
cytosolic PLA2 (26, 27), hepatic lipase (28), guinea pig
pancreatic phospholipase (29), phospholipase B (30),
Campylobacter coli PLA2 (31), and
PS-PLA1 (24), show both PLA and lysophospholipase activity. Among these phospholipases, hepatic lipase (32), guinea pig phospholipase (33), phospholipase B, and PS-PLA1 exhibited
both PLA1 and lysophospholipase activity. Saito et
al. (30) reported that the PLA1 and PLA2
activities of Penicillium notatum phospholipase B are lost
completely by limited proteolysis, whereas its lysophospholipase activity remains unchanged (30), although the protein structures required for each activity have not been characterized yet. Separating lyso-PS-specific lysophospholipase activity from PS-phospholipase A1 is the first example of determining a protein structure
required for lysophospholipase and PLA1 activity.
The lipases (lipoprotein and pancreatic lipase) are reported to be
composed of two domains (the N-terminal and C-terminal domains) (34,
35). PS-PLA1 is also predicted to possess the similar two
domains (24), but in PS-PLA1
C, two-thirds of the C-terminal domain is lost (Fig. 1). From our results (Fig. 3, b and c), we conclude that the N terminus of
PS-PLA1, which is conserved between PS-PLA1 and
PS-PLA1
C (Fig. 1c), carries a structure that
recognizes the serine residues of serine phospholipids (PS and lyso-PS)
and is responsible for heparin binding. We showed in this study that
PS-PLA1
C fails to hydrolyze PS. In addition, our
preliminary experiment shows that when we incorporated lyso-PS as a
substrate of PS-PLA1
C into PC liposomes, its enzymatic
activity was effectively inhibited (data not shown). This indicates
that PS-PLA1
C may not be able to recognize the lipid
surface of PS micelles and that the C terminus of PS-PLA1,
which is missing in PS-PLA1
C, plays an important role in
recognizing diacyl-PS in the lipid bilayers. Several studies have in
fact suggested an important role of the C terminus of lipases in
recognizing lipid surfaces (36, 37); however, as PS-PLA1
does not show any amino acid homology with other lipases in the
C-terminal domain (30), it is also possible that this domain of
PS-PLA1 has another specialized function(s).
It is generally known that a lid is involved in the interfacial
activation (38) and/or substrate recognition (39). The lid of
PS-PLA1 is composed of 12 amino acid residues (Fig. 1), whereas those of many lipases are composed of 22 or 23 residues. This
short lid may not play a role in the interfacial activation, because it
was demonstrated that pancreatic lipase loses the ability to surface
activation when its lid, composed of 23 amino acids, was shortened by
site-directed mutagenesis (38). Rather this short lid may be involved
in substrate recognition. In fact, both PS-PLA1 and
PS-PLA1
C possess the same lid structure.
Expression of PS-PLA1 and PS-PLA1
C in
Human Tissues--
We first purified PS-PLA1 from rat
platelets. In this study, we showed that PS-PLA1 is
expressed in various human tissues. However, human platelets,
leukocytes (Fig. 4), and red blood cells do not express
PS-PLA1 appreciably. We could not detect
PS-PLA1 activity in platelets from rabbits, cattle, or
pigs. Thus, PS-PLA1 is expressed in platelets in a
species-specific manner. It is not yet known whether the alternative
form (PS-PLA1
C) exists in species other than humans,
although the nucleotide sequence of the rat
PS-PLA1 gene around the exon-intron boundary is
identical to that of the human PS-PLA1 gene
(Fig. 2a). Thus, we expect that the alternative form will be
present in the rat and in other species.
Possible Roles of PS-PLA1 and
PS-PLA1
C--
The physiological significance of
PS-PLA1 is not yet known. The chromosomal location of the
human PS-PLA1 gene does not suggest a possible
link to human disease. It is noteworthy, however, that the nucleotide
sequence of human PS-PLA1 is identical to that of the
nmd gene product (40). This gene is predominantly expressed in nonmetastatic human melanoma cell lines, with a lower expression level in metastatic cell lines (40). Any molecular link between PS and
the metastasis of tumor cells is totally unknown at present, but
identifying PS-PLA1 as the nmd gene product
suggests that serine phospholipids (PS or lyso-PS) are involved in the
metastatic process of tumor cells. Calderon et al. (4)
reported that some cancer cells secrete PS, and it impairs macrophage
cytotoxicity by inhibiting the production of nitric oxide from
macrophages. Thus they speculated that cancer cells escape from
macrophage recognition by secreting PS. PS-PLA1 may
hydrolyze such PS and increase the activity of macrophages in
self-defense against tumor cells.
PS-PLA1 mediates three types of reaction to eliminate PS
and 1-acyl-2-lyso-PS and to produce 2-acyl-1-lyso-PS.
PS-PLA1
C, by contrast, has only an ability to eliminate
1-acyl-2-lyso-PS. Once 2-acyl-1-lysophospholipids are produced by the
PLA1 reaction, the fatty acid at the sn-2
position readily migrates to the sn-1 position, which
results in production of 1-acyl-2-lysophospholipids (41).
PS-PLA1
C might eliminate such 1-acyl-2-lyso-PS in
vivo, or it might eliminate 1-acyl-2-lyso-PS, which is produced by
the PLA2 reaction. Because serine phospholipids are
suggested to be involved in various pathophysiological conditions (16),
both PS-PLA1 and PS-PLA1
C might be key
enzymes that regulate the production and elimination of serine
phospholipids. Further study is required to clarify the physiological
functions of PS-PLA1 and PS-PLA1
C.