(Received for publication, October 1, 1996, and in revised form, October 30, 1996)
From the Faculty of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan,
¶ Division of Biomolecular Characterization, Institute of Physical
and Chemical Research (RIKEN), 2-1, Hirosawa, Wako, Saitama 351-01, Japan, and
Faculty of Pharmaceutical Sciences, Osaka University,
1-6, Yamadaoka, Suita, Osaka 565, Japan
Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [3H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.
Membrane phospholipids of activated platelets serve as precursors for second messengers, such as eicosanoids, and also as footholds for blood coagulation. Platelets contain at least three types of phospholipase A; cytosolic phospholipase A2 (cPLA2)1 (1, 2), secretory 14-kDa type II phospholipase A2 (sPLA2) (3), and another serine-phospholipid-selective phospholipase which has not yet been fully characterized (4-6). cPLA2 is involved in the production of eicosanoid by cleaving arachidonic acid at the sn-2 position of phospholipids in various types of cells. sPLA2 is found in inflammatory sites (7, 8) and is considered to be involved in inflammatory response progression and may participate in eicosanoid formation in certain cells, such as vascular endothelial cells, mast cells, neutrophils, hepatocytes, and others (9-13). Thus sPLA2 and cPLA2 are well characterized, and their cDNA and genomic structures are also known (2, 14, 15), whereas no information about the structure of the last enzyme is available.
Like sPLA2 this new member of the phospholipase A family is secreted from activated rat platelets (4); partially purified preparations specifically act on phosphatidylserine (PS) (6) and lysophosphatidylserine (lyso-PS) (4, 5). This activity is inhibited both by diisopropyl fluorophosphate (DFP) and dithiothreitol to release a fatty acid of PS and lyso-PS, respectively (5, 6). On several chromatography columns, PS-hydrolyzing activity co-migrates with lysophospholipase activity detected using lyso-PS as the substrate, suggesting that a single polypeptide may possess both PS-phospholipase A and lyso-PS-lysophospholipase activities. Therefore, throughout the present study we call this enzyme "PS-PLA1."
In this study, we report the purification and cDNA cloning of PS-PLA1. The predicted amino acid sequence did not show any homology to those of phospholipases that have been reported previously, but surprisingly it showed some homology to mammalian lipases such as hepatic lipase (HL), lipoprotein lipase (LPL), and pancreatic lipase (PL). Unlike these "conventional" lipases, the present platelet-derived enzyme did not exhibit appreciable activity to triacylglyceride (TG). Platelets release a novel phospholipase A, upon cell stimulation, that specifically acts on serine-phospholipids and not on any other lipids.
1-Palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine,
1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoinositol,
1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine
1,2-di[14C]oleoyl-sn-glycero-3-phosphocholine,
tri[14C]oleoyl-glycerol, and
1,3-[3H]diisopropyl fluorophosphate were from
Dupont NEN.
1,2-Di[14C]oleoyl-sn-glycero-3-phosphoserine
and
1-[14C]oleoyl-sn-glycero-3-phosphoserine
were prepared as described previously (5, 6).
[-32P]dCTP was the product of Amersham
International (Amersham, UK). PS, phosphatidylethanolamine,
phosphatidylcholine, phosphatidic acid, phosphatidylinositol,
1-acyl-sn-glycero-3-phosphocholine, 1-acyl-sn-glycero-3-phospho-myo-inositol,
and 1,2-diacylglycerol were obtained from Serdary Research
Laboratories (London, UK). 1-Acyl-sn-glycero-3-phosphoserine,
1-acyl-sn-glycero-3-phosphoethanolamine, and
lysophosphatidic acid were from Avanti Polar Lipids (Alabaster, AL). Triolein was the product of Sigma. DEAE-Sepharose
CL-6B, heparin-Sepharose CL-6B, blue Sepharose CL-6B, and Sepharose 4B were bought from Pharmacia Fine Chemicals (Uppsala, Sweden). Other chemicals were purchased from Wako (Osaka, Japan).
1-[1-14C]Palmitoyl-2-acyl-sn-glycero-3-phosphoserine was used to detect PS-PLA1 activity in column chromatography fractions. Phospholipase A1 and lysophospholipase activities were measured as described previously (5, 6). For determination of phospholipid specificity, 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoserine, 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoinositol, 1-palmitoyl-2-[14C] arachidonoyl-sn-glycero-3-phosphatidic acid, and 1-palmitoyl-2-[14C] arachidonoyl-sn-glycero-3-phosphocholine (40 µM each) were incubated with recombinant PS-PLA1 in 100 mM Tris-HCl pH 7.5, 4 mM CaCl2 at 37 °C for 15 min, and the products were analyzed by thin layer chromatography using chloroform/methanol/acetic acid/H2O (25/15/4/2; v/v/v/v). The radioactivities corresponding to each product were detected and analyzed using a BAS 2000 imaging analyzer (Fuji Film, Kanagawa, Japan).
Purification of Phosphatidylserine-specific Phospholipase A1Blood was collected from 200-300 Wistar rats by cardiac puncture, and platelet-rich plasma was prepared as described previously (5). The platelets were activated by incubating with 2 units/ml thrombin and 2 mM CaCl2 for 20 min at 37 °C. Then, the plasma was centrifuged at 1,500 × g for 10 min at 4 °C, and the supernatant was used as the enzyme source.
The supernatant was loaded onto a DEAE-Sepharose CL-6B column (30 ml), which had been equilibrated with 100 mM NaCl, 10 mM Tris-HCl, pH 7.4. The flow-through fraction was then loaded onto a heparin-Sepharose CL-6B column (10 ml), which had been equilibrated with 100 mM NaCl, 10 mM Tris-HCl, pH 7.4. The column was washed thoroughly with this buffer and eluted with a linear gradient of NaCl (0.1-1.0 M). The pooled fractions containing lysophospholipase activity were applied to a blue Sepharose CL-6B column (2 ml). After the column was washed with 500 mM NaCl, 10 mM Tris-HCl, pH 7.4, the enzyme was eluted by changing the buffer to 1 M NaCl, 50 mM Tris-HCl, pH 9.0.
DFP LabelingAliquots of various fractions that contained PS-PLA1 activity were mixed with [3H]DFP (5 µCi, 0.58 nmol) and incubated for 15 min at room temperature. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography were carried out as described previously (16).
Amino Acid Sequence Analysis of PS-PLA1The N-terminal amino acid sequence was determined by the method of Matsudaira (17). Briefly, the partially purified protein (the active fractions from the blue column) was concentrated, and the protein was separated by SDS-PAGE. After blotting onto a polyvinylidene difluoride membrane, the protein band was visualized by Coomassie staining, cut out, and subjected to automated Edman degradation using an ABI model 477A protein sequencer connected on-line to an ABI model 120A phenylthiohydantoin analyzer (Perkin-Elmer).
For determination of internal amino acid sequences, the protein was S-carboxymethylated and separated by SDS-PAGE, and in situ fragmentation either by CNBr treatment or by digestion with lysyl endopeptidase was performed. The resulting peptide fragments were separated by reverse-phase high performance liquid chromatography using a gradient of 0-64% acetonitrile in 0.1% trifluoroacetic acid. Alternatively, the peptides were separated by SDS-PAGE using a 10-24% acrylamide gel and blotted to a polyvinylidene difluoride membrane. The isolated peptides were sequenced in an ABI model 477A protein sequencer.
cDNA Cloning of PS-PLA1A rat megakaryocyte
gt11 phage cDNA library (18) was used for screening. The
cDNA fragment that contained internal amino acid sequences was
amplified by the polymerase chain reaction (PCR) using the
megakaryocyte
gt11 phage cDNA library as the template, and the
following degenerated oligonucleotide primers; GTICCICCICCIACNCARCC and
ACICGIGADATRTGGAA, based on the VPPPTQP and FHISSRV sequences of the
two peptides (N-terminal and Lys-end-2) (see Table II), respectively.
The resulting PCR fragments were subcloned into a pT7Blue T-vector
(Novagen, Madison, WI), and the nucleotide sequences were determined. A
DNA fragment which contains internal amino acid sequences was chosen
and used for identifying
phages which contained same sequence as
the DNA fragments by plaque hybridization. The insert DNA was cut off by digestion from the phage DNA with EcoRI and was subcloned
into the EcoRI site of the pBluescript II SK phagemid vector
(Stratagene, La Jolla, CA). After the restriction enzyme map (Fig.
3A) was determined, the nucleotide sequence was determined
by DNA sequencing. 5
-RACE was performed using a Marathon cDNA
amplification kit (Clontech, Palo Alto, CA) with rat platelet total RNA
as the template for first strand cDNA synthesis according to the
manufacturer's protocol. The antisense oligonucleotide
TTCCGGATGTCACTGTCCTC (nucleotides 205-224 of PS-PLA1, see
Fig. 3B) was used as a primer for first strand synthesis.
|
Expression of PS-PLA1 in Sf9 Cells
cDNA
encoding the coding region of PS-PLA1 (nucleotide positions
13-1729) was inserted into the SmaI and EcoRI
sites of a baculovirus transfer vector pVL1393 (PharMingen, San Diego,
CA). The plasmid obtained was designated PS-PLA1-pVL1393. A
recombinant virus was prepared using the BaculoGold system (PharMingen)
according to the manufacturer's protocol. Cells (6 × 105 cells/ml) were mixed with recombinant or wild-type
Autographa californica nuclear polyhedrosis virus (AcNPV)
(multiplicity of infection = 10) and incubated for 96 h at
27 °C. Phospholipase A1 and lysophospholipase activities
in the cell culture supernatant were determined as described above.
[14C]PS (2 µM) or [14C]TG (2 µM) was incubated with the enzyme in 4 mM CaCl2, 100 mM Tris-HCl, pH 7.5, at 37 °C for 15 min in the presence of 300 µM Triton X-100. After extraction from the mixture by Bligh and Dyer's method, the release of lyso-PS and diglyceride was measured by TLC using chloroform/methanol/acetic acid/H2O (25/15/4/2; v/v/v/v) and hexane/ether/acetic acid (70/30/1; v/v/v), respectively. Radioactivity was detected with BAS 2000 imaging analyzer. In some experiments, [14C]PS (2 µM) was mixed with nonlabeled TG (2 µM) in the presence of 300 µM Triton X-100. Similarly, [14C]TG (2 µM) was mixed with nonlabeled PS (2 µM) in the presence of 300 µM Triton X-100. The lipid mixture was then incubated with the enzyme in 4 mM CaCl2, 100 mM Tris-HCl, pH 7.5, at 37 °C for 15 min.
Other Analytical MethodsProtein concentrations were determined using the BCA protein assay reagent (Pierce). SDS-PAGE was performed by the method of Laemmli. DNA sequencing was performed using an ALFred DNA sequencer with an AutoCycle Sequencing Kit (Pharmacia Biotech Inc.).
As rat platelets released both
PS-selective phospholipase A1 and lyso-PS-selective
lysophospholipase activities upon cell activation, the extracellular
medium from thrombin-activated platelet cultures was used as the enzyme
source. Lyso-PS was used as a substrate for assessing enzyme activity
throughout the present study. First the medium was subjected to
DEAE-Sepharose column chromatography. The enzyme passed through the
column, whereas 40% of the total protein was absorbed. Next the
flow-through fraction was applied to a heparin-Sepharose column, and
the enzyme activities were eluted with linear gradient of NaCl. The
activities were eluted in fractions at about 250 mM NaCl.
The pooled fractions were then applied to a blue Sepharose column, and
the activities were eluted by 1 M NaCl. Typical elution
profiles of the activities from the heparin-Sepharose and blue
Sepharose columns, and the SDS-PAGE pattern of the fractions collected
from the heparin-Sepharose and blue Sepharose columns are shown in Fig.
1. A major 55-kDa protein co-migrated with the
lysophospholipase activity on the blue Sepharose column (Fig.
1B). This 55-kDa protein also co-migrated with
lysophospholipase activity on the heparin-Sepharose column (Fig.
1A) and on other types of chromatography columns that we tested, such as a gel filtration column and a CM-Sepharose column (data
not shown). The purification procedure is summarized in Table
I. The overall purification was about 1800-fold, and the recovery was 31%.
|
DFP inhibited
both PS-PLA1 and lyso-PS-lysophospholipase activities in
the purified preparation. To further confirm that the 55-kDa protein
was the enzyme itself, we next performed a
[3H]DFP-labeling experiment. DFP inhibits the activities
of various serine esterases, including lipases, by reacting covalently
with an active serine residue of these enzymes. Fractions positive for
the enzyme activity that were obtained during the purification procedure were then incubated with [3H]DFP, and
incorporation of the radioisotope was analyzed by SDS-PAGE and
fluorography. As shown in Fig. 2, the purified 55-kDa
polypeptide did incorporate [3H]DFP, confirming that it
possesses an active serine residue. Labeling of the 55-kDa polypeptide
with [3H]DFP was seen in the fraction obtained after the
heparin-Sepharose and blue Sepharose chromatography (Fig. 2). Other
bands which were detected in these partially purified preparations were
not components of the enzyme, because they did not co-migrate with the
enzyme activities on several chromatography columns (data not shown).
Thus we conclude that the 55-kDa protein is the enzyme itself.
cDNA Cloning of PS-PLA1
We next determined
the N-terminal and internal amino acid sequences of purified
PS-PLA1. Table II shows the sequences of the peptides obtained. On the basis of these sequences, degenerated oligonucleotides were synthesized and used in a series of PCRs with a
rat megakaryocyte gt11 cDNA library as a template. One set of
primers yielded an amplified product containing sequences corresponding
to several peptides (Fig. 3A). Therefore we
concluded that this amplified product was generated from the
PS-PLA1 cDNA and utilized it to screen a rat
megakaryocyte
gt11 cDNA library. As a result two
clones were
obtained and sequenced (Fig. 3A). However, these two clones
did not contain the entire coding region, so we amplified cDNA
corresponding to the 5
-region by 5
-RACE. The nucleotide sequence of
the PS-PLA1 cDNA is shown in Fig. 3B. The
deduced amino acid sequence contains all amino acid sequences obtained
from N-terminal and internal amino acid sequence analysis. It contains
an open reading frame of 1368 base pairs which encodes a 456-amino acid
polypeptide. A hydrophobic sequence, composed of 24 amino acid
residues, is present at the N-terminal of the deduced amino acid
sequence. This hydrophobic sequence is probably a signal sequence
because Gly25 is an N-terminal amino acid of purified
protein (Table II). The deduced amino acid sequence contains three
possible sites for N-linked glycosylation, a putative active
serine (in a Gly-X-Ser-X-Gly motif), and 16 cysteine residues. The presence of the motif of the active serine
residue explains the inhibition of the activity of this enzyme by the
serine esterase inhibitor DFP (5).
To confirm that the
cDNA obtained encoded PS-PLA1, a recombinant
baculovirus was prepared and used to infect Sf9 insect cells. As shown
in Fig. 4A, the culture medium of Sf9 cells
infected with the recombinant baculovirus exhibited appreciable
lysophospholipase activity, whereas the culture medium obtained with
wild-type virus did not. The medium of cells infected with the
recombinant virus also showed PS-hydrolyzing activity (Fig.
4B), confirming that both lysophospholipase and
PS-hydrolyzing activities were mediated by the same protein. The
activity was mostly recovered from the medium, suggesting that the
enzyme was spontaneously secreted from Sf9 cells. In platelets the
enzyme might be stored in granules and secreted upon cell
activation.
Sequence Analysis
We searched the protein data base for
similar sequences using the BLASTN program (19). LPL, HL, and PL of
various species such as human, rat, and mouse showed significant
homology with the enzyme (Fig. 5A). The
enzyme showed 30.8, 31.1, and 29.1% identity to rat LPL (20), HL (21),
and PL (22), respectively. Each of these lipases (LPL, HL, and PL) is
composed of two domains (the N- and C-terminal domains) (23, 24). As
shown in Fig. 5A, the homologous regions (amino acids 1-280
in PS-PLA1) are in the first half of the molecule, which
corresponds to the N-terminal domain of lipases. In particular, amino
acid residues surrounding Ser142, Asp166, and
His236 in PS-PLA1 are well conserved in these
three lipases and may form a "catalytic triad" in
PS-PLA1, because the amino acids corresponding to these
three amino acids in LPL, HL, and PL make up catalytic triads (25). In
addition, six cysteine residues (amino acid positions 221, 234, 258, 269, 272, and 280 in PS-PLA1) are completely conserved in
LPL, HL, and PL (Fig. 5A). A potential
N-glycosylation site (Asn55 in
PS-PLA1) is also conserved in LPL and HL. It has been
reported that glycosylation at the conserved asparagine of LPL and HL
is essential for the synthesis of fully active enzymes (26, 27). The
latter half of the PS-PLA1 molecule (amino acids 281-426) did not show significant homology with LPL, HL, or PL (Fig.
5A). Lipase lids exist in LPL and HL, and are surrounded by
two cysteine residues (216 and 239 in LPL, and 239 and 262 in HL) (24).
These lids are reported to be involved in determining the substrate specificity of the two enzymes (28). In PS-PLA1 the region
corresponding to the lipase lids (amino acids 222-233, the putative
lid of PS-PLA1) is composed of 12 amino acid residues,
whereas the lids of both LPL and HL are composed of 22 amino acid
residues. Thus the putative lid of PS-PLA1 is very short.
The phylogenetic relationship between the lipase family and
PS-PLA1 is shown in Fig. 5B. This shows that
PS-PLA1 is more closely related to LPL and HL than PL.
Substrate Specificity of PS-PLA1
From 500 ml of culture medium of Sf9 cells infected with recombinant virus we purified about 160 µg of recombinant enzyme by the same method as that used for rat platelet enzyme. This recombinant enzyme exhibited a smaller molecular mass (50 kDa) on SDS-PAGE analysis compared with enzyme derived from rat platelets (data not shown). The specific activity of recombinant enzyme toward PS was 2.6 µmol/min/mg, whereas those toward phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and phosphatidylinositol were less than 0.1 µmol/min/mg (Table III). The substrate specificity of the platelet enzyme was the same as that of the recombinant enzyme (data not shown).
|
Next we examined whether the enzyme hydrolyzed TG, because the
structure of the enzyme resembles the lipases (Fig. 5A). No appreciable hydrolysis was observed against triolein in the Triton X-100 micelle system, whereas PS was hydrolyzed under the same conditions (Fig. 6, lanes 1 and
2). Hydrolysis of triolein was not detected even under the
conditions in which the same amount of lipids was introduced in the
assay (Fig. 6, lanes 3 and 4). Thus
PS-PLA1 cannot digest TG in spite of its similarity in
structure to the mammalian lipases.
PS-PLA1 releases fatty acid from 1-acyl-lyso-PS, so it can
cleave the fatty acid of lyso-PS at the sn-1 position. To
test whether PS-PLA1 has phospholipase B activity toward
PS, the ability of PS-PLA1 to cleave a fatty acid at the
sn-2 position was investigated using
1-palmitoyl-2-[14C]arachidonoyl-PS as the substrate. As
shown in Fig. 7 radioactivity was not detected at the
TLC migration position of free fatty acid, but was detected at the
position of lyso-PS. Thus the acyl chain at the sn-2
position in PS is not hydrolyzed by PS-PLA1. When 1,2-di[14C]oleoyl-PS was used as the substrate for a
PS-PLA1 assay, radioactivity was detected in spots
corresponding to both free fatty acid and lyso-PS (data not shown).
These results suggest that PS-PLA1 releases fatty acid from
the sn-1 position in PS and lyso-PS.
In this study we purified PS-PLA1 that was secreted from activated platelets and established its primary structure by cDNA cloning. The primary structure of the enzyme did not exhibit homology to any phospholipase A2 or lysophospholipase that has been reported previously (type I PLA2 (29), type II PLA2 (14), cPLA2 (2), hepatic lysophospholipase (30), Escherichia coli lysophospholipase (31), human eosinophil lysophospholipase (32), and rat pancreas lysophospholipase (33)). Unexpectedly PS-PLA1 resembles some mammalian lipases (LPL, HL, and PL) although it did not show any detectable lipase activity.
Substrate Specificity of PS-PLA1The recombinant form of PS-PLA1 hydrolyzed PS and lyso-PS at the sn-1 position of the glycerol backbone to release a fatty acid (Fig. 7), but did not hydrolyze other phospholipids (Table III). A previous study using partially purified enzyme showed that the enzyme hydrolyzed lysophosphatidylserine but did not hydrolyze other lysophospholipids such as lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidic acid (5). The enzyme did not hydrolyze TG, even though it did hydrolyze PS under the same conditions (Fig. 6), and porcine pancreatic lipase hydrolyzed TG (data not shown). Thus PS-PLA1 cannot hydrolyze TG, even though it shares some sequence homology with mammalian lipases. HL is known to possess both lipase and phospholipase activities. In addition, a phospholipase A1 from hornets, which has a sequence similarity of about 40% with mammalian lipases, has phospholipase A1 activity (using phosphatidylcholine as the substrate) and weak lipase activity (34). The substrate specificity of PS-PLA1 is different from that of lipases because it cannot react with TG, but does recognize the glycerophosphoserine structure and hydrolyzes a fatty acyl residue bound at the sn-1 position of the glycerol backbone.
Which part of the molecular structure of PS-PLA1 determines
its substrate specificity? Under aqueous conditions, lipases are inactive, as the "catalytic triad" is buried beneath a short
amphipathic -helix (the "lid") (23). Compared with the lid amino
acid sequences of other lipases, the putative lid of
PS-PLA1 is extremely short (Fig. 5A). Rat LPL
and HL have lids composed of 22 amino acids between two conserved
cysteine residues, whereas the corresponding region in
PS-PLA1 is composed of only 12 amino acid residues. It has
been reported that the substrate specificities of LPL and HL are
determined by the lids of the molecules (28). Guinea pig PL has a short
lid and exhibits both phospholipase A1 and lipase
activities (35), and hornet phospholipase A1, which has only 7 amino acids in the corresponding region, has weak lipase activity (34). Thus the "phospholipase A1 activity" of
lipase family molecules may depend on the length and amino acid
sequence of the lid.
The mechanism by which PS-PLA1 specifically recognizes
serine-phospholipids is not known at present. A cluster of positively charged amino acids (Lys and Arg) is present in the sequence of PS-PLA1 (348-353). It is interesting that the amino acid
sequences around this cluster
(VEN
EKRK
DT) resemble the
PS-binding motif found in protein kinase C and PS decarboxylase
(FXFXLKXXXXKXR) (36). Amino
acid sequences of lipases around these regions are full of variety.
This region may be responsible for determining the substrate
specificity of PS-PLA1. Mutation studies will test this hypothesis.
Of the known mammalian phospholipase As, rat platelet PS-PLA1 is the only one that is a glycoprotein. Three potential N-glycosylation sites are present in the deduced amino acid sequence of PS-PLA1. The idea that PS-PLA1 is a glycoprotein was confirmed by two additional observations: 1) in our preliminary experiments, PS-PLA1 and lyso-PS lysophospholipase activities were retained on concanavalin A-, WGA-, and RCA-Sepharose columns and were eluted by the hapten sugars of each lectin; and 2) the calculated molecular mass of PS-PLA1 is 47 kDa, whereas the purified protein had a molecular mass of 55 kDa by SDS-PAGE analysis. This difference may be due to the addition of an asparagine-linked sugar moiety because treatment of the purified protein with N-glycosidase F, which removes N-linked sugar moieties, reduced the molecular mass of platelet PS-PLA1 to 48 kDa (data not shown). The molecular mass of the recombinant protein, produced in a baculovirus system, was 50 kDa. The difference in molecular mass observed between rat platelet and recombinant PS-PLA1 may be due to differences in the sugar structure because the molecular mass of recombinant PS-PLA1 after N-glycosidase F treatment was also 48 kDa. A strict carbohydrate structure seems not to be required for PS-PLA1 activity. This fact is in contrast with LPL and HL, since it has been reported that an N-linked sugar on LPL and HL at the N-terminal (amino acid 75) is necessary for enzyme activity (26, 27). In addition, PS-PLA1 has no homology with phospholipase B of Penicillium notatum (37) or Saccharomyces cerevisiae (38), both of which are glycoproteins.
Secretion of PS-PLA1 from Activated PlateletsPS-PLA1 was released from rat platelets
when the cells were stimulated with thrombin. The enzyme is also
released in response to other stimuli such as ADP and A23187 (5). In
unstimulated platelets PS-PLA1 may be stored in
-granules, since type II PLA2, which is known to be
present in
-granules, is released from rat platelets in a similar
manner to PS-PLA1 upon stimulation with thrombin (5). The
mechanism of PS-PLA1 sorting to secretory granules is not
known at present. The N-terminal hydrophobic sequence of
PS-PLA1, which consisted of 24 amino acids, may be a signal sequence because Gly25 is an N-terminal amino acid of the
purified enzyme (Table II). According to the PSORT program, which
predicts protein sorting from the primary amino acid sequence (39), the
24 hydrophobic amino acid sequence of PS-PLA1 does not show
features typical of the signal sequences of "secretory" proteins.
The enzyme was secreted from infected Sf9 cells, supporting the fact
that the signal observed in the present enzyme functions as a signal
sequence.
The biological function of PS-PLA1 is unknown at present. It is well known that membrane phospholipids of activated platelets serve as precursors for second messengers such as eicosanoids and also as footholds for blood coagulation. PS-PLA1 is released from activated platelets. Under the conditions, 2-acyl lyso-PS may accumulate on the membrane, since PS-PLA1 attacks only the fatty acyl residue at the sn-1 position of serine-phospholipids. PS and lyso-PS may play important roles in either intracellular signal transduction or intercellular blood coagulation processes. PS-PLA1 may be involved in the regulation of these processes through control of the concentration of PS or lyso-PS.
We thank Drs. Osamu Kuge, Shuntaro Hara, and Hideki Adachi for technical advice for PCR cloning and Dr. Yuji Sato, Atsushi Matsuzawa, Tomoko Yoshizumi, Kotaro Hirota, Masako Ogawa, Tomoya Adachi, Koji Bando, Kazuhiro Nomura, and Miki Watanabe for collection of rat platelets. We greatly appreciate Drs. Nakahara and Okamoto (Fujisawa Pharmaceutical Co. Ltd.) for generous provision of rats.