From the Department of Chemistry, University of
Illinois, Chicago, Illinois 60607 and the § Department of
Medicine, University of Chicago, Chicago, Illinois 60307
Received for publication, May 28, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Human group V phospholipase A2
(hVPLA2) has been shown to have high activity to elicit
leukotriene production in human neutrophils (Han, S. K., Kim,
K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999)
J. Biol. Chem. 274, 11881-11888). To determine the
mechanism by which hVPLA2 interacts with cell membranes to
induce leukotriene formation, we mutated surface cationic residues and
a catalytic residue of hVPLA2 and measured the interactions
of mutants with model membranes, immobilized heparin, and human
neutrophils. These studies showed that cationic residues,
Lys7, Lys11, and Arg34, constitute
a part of the interfacial binding surface of hVPLA2, which
accounts for its moderate preference for anionic membranes. Additionally, hVPLA2 binds heparin with high affinity and
has a well defined heparin-binding site. The site is composed of
Arg100, Lys101, Lys107,
Arg108, and Arg111, and is spatially distinct
from its interfacial binding surface. Importantly, the activities of
the mutants to hydrolyze cell membrane phospholipids and induce
leukotriene biosynthesis, when enzymes were added exogenously to
neutrophils, correlated with their activities on phosphatidylcholine
membranes but not with their affinities for anionic membranes and
heparin. These results indicate that hVPLA2 acts directly
on the outer plasma membranes of neutrophils to release fatty acids and
lysophospholipids. Further studies suggest that products of
hVPLA2 hydrolysis trigger the cellular leukotriene
production by activating cellular enzymes involved in leukotriene
formation. Finally, the temporal and spatial resolution of exogenously
added hVPLA2 and mutants suggests that binding to cell
surface heparan sulfate proteoglycans is important for the
internalization and clearance of cell surface-bound
hVPLA2.
Phospholipases A2
(PLA2)1 catalyze
the hydrolysis of membrane phospholipids, the products of which can be
transformed into potent inflammatory lipid mediators,
platelet-activating factor, and eicosanoids that include
prostaglandins, thromboxanes, leukotrienes, and lipoxins. Multiple
forms of PLA2s have been found in mammalian tissues
(1), including several forms (groups Ib, IIa, IIc, IId,
IIe, IIf, V, and X) of secretory PLA2s (sPLA2)
and intracellular PLA2s (e.g. groups IV and VI).
Recent cell studies indicated that sPLA2 works in concert
with group IV cytosolic PLA2 to induce immediate and
delayed eicosanoid formation (2, 3). At present, however, the identity
of sPLA2 involved in eicosanoid biosynthesis, the temporal
and spatial sequences of its mobilization during inflammatory cell
activation, and the mechanism by which it interacts with cell membranes
to induce cellular eicosanoid formation are not fully understood.
Recently, mounting evidence has pointed to the involvement of group V
PLA2 in eicosanoid formation in various mammalian cells
(4-8) but the mechanisms of its eicosanoid-inducing activities have
not been elucidated. In particular, the mechanism whereby group V
PLA2 interacts with cell membranes remains unclear. There
are at least three possible mechanisms for interaction of sPLA2 with cells (see Fig. 1). In the simplest mechanism,
sPLA2 would act directly on membrane phospholipids to
release fatty acids and lysophospholipids, which in turn induce
eicosanoid release. However, many mammalian sPLA2s, most
notably group Ib (9) and IIa PLA2s (10), strongly prefer
anionic membranes to zwitterionic ones, and consequently have low
affinity for and activity on intact mammalian cells, the outer plasma
membranes of which are mainly composed of zwitterionic
phosphatidylcholine (PC) and sphingomyelin. For this reason, indirect
mechanisms, involving anionic cell surface heparan sulfate
proteoglycans (HSPG) (3, 11-14) and protein receptors (15), have been
proposed (see Fig. 1). However, direct involvement of HSPG and receptors in the actions of sPLA2
on cells remains controversial (16). To elucidate the origin of high activity of group V PLA2 to release fatty acids and
eicosanoids from mammalian cells, we fully characterized the enzymatic
and membrane-binding properties of recombinant human group V
PLA2 (hVPLA2) (17, 18). These studies showed
that hVPLA2 could bind and hydrolyze PC membranes and the
outer plasma membranes of mammalian cells much more efficiently than
other sPLA2s, including human group IIa PLA2
(hIIaPLA2). Additionally, a structure-function analysis of
hVPLA2 revealed good correlation between its affinity for
PC membranes and its activity on mammalian cells, suggesting that
hVPLA2 could directly bind to the outer plasma membranes of
mammalian cells to hydrolyze phospholipids and elicit eicosanoid formation (18). However, it was also reported that rat group V
PLA2 binds to human embryonic kidney (HEK) 293 cells via
HSPG (3). Indeed, group V PLA2s, including
hVPLA2, contain a cluster of cationic residues in the
C-terminal region (see Fig. 2), which is
involved in HSPG binding for group IIa PLA2s (16). In
addition, hVPLA2 has a few cationic residues on its
putative interfacial binding surface and modestly (~4-fold) prefers
anionic membranes to zwitterionic membranes (18). These findings
suggested that the cell binding of group V PLA2 might
involve interactions with anionic surfaces, including cell surface
HSPG. To determine the exact mechanism by which hVPLA2
interacts with mammalian cells and elucidate the potential involvement
of HSPG binding in its cell activities, we mutated surface cationic
residues and a catalytic residue, and measured the interactions of
mutants with model membranes, immobilized heparin, and human
neutrophils. Results provide new insights into the temporal and spatial
sequences of events involved in hVPLA2-mediated cellular
eicosanoid biosynthesis.
Materials--
1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoethanolamine
(pyrene-PE) was purchased from Molecular Probes (Eugene, OR).
1,2-sn-Dioleoylglycerol,
1,2-dihexanoyl-sn-glycero-3-phosphoglycerol (diC6PE), and
1-palmitoyl-2-hyrdoxy-sn-glycero-3-phosphocholine were from
Avanti Polar Lipids (Alabaster, AL).
1,2-Bis[12-(lipoyloxy)-dodecanoyl]-sn-glycero-3-phosphoglycerol (BLPG) was prepared as described elsewhere (19, 20).
1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) was from Sigma. Phospholipid concentrations were determined by
phosphate analysis (21). [3H]Arachidonic acid (AA) was
purchased from American Radiochemical Co. (St. Louis, MO).
[14C]Oleic acid (OA) and
1-stearoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine
([14C]SAPC) (55 mCi/mmol) were from Amersham Pharmacia
Biotech. Styrene-divinylbenzene beads (5.2 ± 0.3 µm diameter)
were purchased from Seradyn (Indianapolis, IN). Fatty acid-free bovine
serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). All restriction
enzymes, T4 ligase, and T4 polynucleotide kinase were obtained from
Roche Molecular Biochemicals. Oligonucleotides were purchased from
Integrated DNA Technologies (Coralville, IA) and used without further
purification. Recombinant hIIaPLA2 was prepared as
described (10).
Mutagenesis, Expression, and Purification of
hVPLA2--
Mutagenesis was performed using the Sculptor
in vitro mutagenesis kit from Amersham Pharmacia Biotech and
a phagemid DNA prepared from the pSK vector in the presence of helper
phage R408 as described previously (22). Wild type and mutant proteins
were expressed in Escherichia coli, refolded, and purified
as described previously (17). Purified proteins were lyophilized and
stored at Kinetic Measurements--
PLA2-catalyzed hydrolysis
of polymerized mixed liposomes was carried out at 37 °C in 2 ml of
10 mM HEPES buffer, pH 7.4, containing 0.1 µM
pyrene-PE (1 mol %) inserted in 9.9 µM BLPG, 2 µM BSA, 0.16 M NaCl, and 10 mM
CaCl2 (19, 20). The progress of hydrolysis was monitored as
an increase in fluorescence emission at 378 nm using a Hitachi F4500
fluorescence spectrometer with the excitation wavelength set at 345 nm.
Spectral bandwidth was set at 5 nm for both excitation and emission.
Values of kcat*/Km* were determined from reaction progress curves as described previously (10).
The PLA2-catalyzed hydrolysis of diC6PE
monomers was performed with 0.5 mM phospholipid, 0.16 M NaCl, and 10 mM CaCl2. The time course of phospholipid hydrolysis was monitored with a
computer-controlled Metrom pH-stat (Brinkmann) in a thermostated
vessel. Under these conditions, the hydrolysis of diC6PE
followed the first-order kinetics because the substrate concentrations
remained lower than apparent Km. Thus, the values of
apparent second-order rate constant,
(kcat/Km)app,
were calculated by dividing by the enzyme concentration the
pseudo-first-order rate constants determined from the nonlinear
least-squares analysis of reaction progress curves. Activity of
PLA2 on zwitterionic vesicles was assayed by measuring the
initial rate of [14C]SAPC hydrolysis. Typically, 20 µl
of [14C]SAPC solution in chloroform was dried in a glass
vial with N2 and hydrated in 800 µl of 10 mM
HEPES buffer, pH 8.0, containing 0.16 M NaCl and 10 mM CaCl2. After vortexing, the lipid suspension was sonicated for about 10 s, frozen in ethanol-dry ice bath, and
sonicated again for ~1 min to afford a homogenous small unilamellar vesicle solution. For assay, 50-µl aliquots of
[14C]SAPC vesicles (~10 µM) solutions
containing 15 µM BSA were placed in Eppendorf tubes.
Reactions were started by addition of enzyme to a final concentration
of 10-20 nM and quenched by adding 370 µl of ice-cold
chloroform/methanol/HCl (2:1:0.01, v/v/v) solution after a given period
of incubation at room temperature. Subsequently, 460 µl of
chloroform/methanol/H2O (1:2:0.8, v/v/v), 240 µl of chloroform, and 240 µl of H2O were added and each
reaction mixture was vortexed to extract the lipids into the organic
phase. Liberated [14C]AA was separated from the reaction
mixtures on small silica gel columns using petroleum ether/ether/acetic
acid (70:30:1, v/v/v) as an eluent. Solvents collected in scintillation
vials were evaporated with N2 and replaced by 4 ml of
scintillation mixture (Sigma), and the radioactivity was measured by
liquid scintillation counting.
Binding of PLA2 to Phospholipid-coated Beads and
Vesicles--
The binding affinity of PLA2 for
sucrose-loaded polymerized BLPG liposomes was determined at 25 °C as
described previously (10). The binding assay solution contained 100 µM phospholipid and varying concentrations of
PLA2 in 10 mM Tris-HCl buffer, pH 7.4, containing 0.145 M NaCl, 10 mM
CaCl2, and 1 µM BSA. For the determination of
PC membrane binding affinity, PC-coated styrene-divinylbenzene beads,
which can be rapidly and completely separated from the solution by low
speed centrifugation, were used instead of PC vesicles that typically
have low pelleting efficiency (23). Binding measurements with
DHPC-coated beads were performed as described previously (24). The
binding assay solution contained 100-150 µM phospholipid
coated on beads and varying concentrations of PLA2 in 10 mM Tris-HCl buffer, pH 7.4, containing 0.16 M
NaCl, 10 mM CaCl2, and 1 µM BSA.
For both binding measurements, the concentration of free enzyme was
determined by PLA2 activity assays using either
pyrene-PE/BLPG polymerized mixed liposomes or
[14C]SAPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/1,2-sn-dioleoylglycerol (10:9:1) vesicles as substrate, from which the bound enzyme
concentration ([E]b) was calculated. Values of
n and Kd were determined by nonlinear
least-squares analysis of the [E]b versus
[E]o plot using Equation 1.
Heparin Binding--
Heparin-binding affinity of W79A and other
mutants was measured using a HiTrap heparin-Sepharose column (Amersham
Pharmacia Biotech) connected to an Äkta Fast Protein Liquid
Chromatography system (Amersham Pharmacia Biotech). Typically, ~40
µg of hVPLA2 dissolved in 20 mM Tris-HCl, pH
7.4, was loaded onto an 1-ml column that was equilibrated with the same
buffer solution at 4 °C. The column was then eluted at 1 ml/min with
a linear gradient of 0-0.7 M KCl in the same buffer. The
enzyme fractions were monitored by a PLA2 activity assay
using
[14C]SAPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/1,2-sn-dioleoylglycerol (10:9:1) vesicles as substrate as described above. The concentration of
KCl that gives rise to a protein peak was determined for each protein
as an average of duplicate measurements.
Preparation of Neutrophils--
Human neutrophils were prepared
from heparinized venous blood collected from medication-free donors
according to the method by Hansel et al. (25) with
modifications. Briefly, 30 ml of heparinized blood was diluted with the
equal volume of calcium-free Hanks' balanced salt solution (HBSS),
layered over 15 ml of 1.089 g/ml Percoll and centrifuged for 20 min at
900 × g. The supernatant and the mononuclear cells at
the interface were aspirated carefully, and the inside wall of the tube
was wiped with sterile gauze to remove mononuclear cells attached to
the wall. To the pellet of neutrophils and erythrocytes was added 20 ml
of ice-cold water and the suspension was mixed gently for 30 s,
after which 20 ml of 2× HBSS was added. If the erythrocytes remained,
then the procedure was repeated. After the lysis of erythrocytes,
neutrophils were washed once in HBSS plus 0.2% BSA, and the total cell
count was determined with a Coulter counter. The resulting cell
population consisted of >95% neutrophils, as estimated by
differential counts of Wright-Giemsa-stained cytospin preparations.
Fatty Acid Release and Eicosanoid Production from
Neutrophils--
Radiolabeling of neutrophils was achieved by
incubating the cells (107) with 0.5 Ci/ml
[3H]AA for 90 min at 37 °C. Unincorporated
[3H]AA was removed by washing the cells with HBSS plus
0.2% BSA. Double labeling of neutrophils with [3H]AA
(0.5 Ci/ml) and [14C]OA (0.5 Ci/ml) was performed in the
same manner. The labeled cells were resuspended in HBSS plus 0.2% BSA
(106 cells/90 µl) and incubated with 0.1 µM
PLA2 at 37 °C for a given period (30 min for routine
assays). The reaction was quenched by centrifugation, and the
supernatants were removed and their radioactivity (3H and
14C) was measured by two-channel liquid scintillation
counting. For the measurements of leukotriene production from
neutrophils, cells (1 × 106 cells/ml) were incubated
at 37 °C in 250 µl of HBSS containing CaCl2 (1.2 mM) and 0.1 µM PLA2. Control
cells were treated with HBSS. Thereafter cells were centrifuged at
8000 g for 2 min. Leukotriene B4 (LTB4)
release was determined using an enzyme immunoassay kit from Cayman
Chemical Co. (Ann Arbor, MI). Typically, LTB4 secretion reached a maximal value within 20 min of incubation under our experimental conditions. The maximal value for each incubation mixture
was then corrected for a background signal from control cells.
Western Blotting Analysis of hVPLA2-treated Human
Neutrophils--
Neutrophils (2 × 106 cells per
experiment) were treated with 0.3 µM hVPLA2
for various periods, and the incubation was quenched by centrifugation
at 12,000 × g for 10 s. After repeatedly (more than three) washing, the pellets with HBSS containing 0.5 M
NaCl, the pellet was then lysed in 70 µl of lysis buffer (20 mM Tris-HCl, 30 mM
Na4P2O7, 50 mM NaF, 40 mM NaCl, 5 mM EDTA, pH 7.4) containing 1%
Nonidet P-40, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 2 mM
Na3VO4, and 0.5% deoxycholic acid. After 10 min on ice, the cell lysates were centrifuged at 12,000 × g for 20 min to remove cell debris. The supernatants were
then mixed with 14 µl of gel loading buffer (0.125 M
Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% SDS, 0.005% bromphenol
blue), and the mixtures were boiled for 5 min. The samples were
subjected to SDS-polyacrylamide gel electrophoresis under reducing
condition, using 16% acrylamide gels. The electrotransfer of proteins
from the gels to polyvinylidene fluoride membrane was achieved using a
semidry system (200 mA, 120 min). The membrane was blocked with 2% BSA
for 60 min, then incubated with 2 µg/ml of anti-hVPLA2
monoclonal antibody 3G1 (26) diluted in Tris-buffered saline plus
0.05% Tween 20 (TBS-T) overnight. The membranes were washed three
times for 20 min with TBS-T. Goat anti-mouse IgG conjugated with
horseradish peroxidase was diluted 1:3000 in TBS-T and incubated with
polyvinylidene fluoride membrane for 60 min. The membrane was again
washed three times with TBS-T and assayed with an ECL chemiluminescence
system (Amersham Pharmacia Biotech).
Confocal Microscopy Imaging--
Neutrophils were re-suspended
in HBSS at a density of 5 × 105 cells/ml. 300 µl of
neutrophils in HBSS were placed into each of eight wells on a sterile
NuncTM chambered cover glass and incubated at 37 °C with 5%
CO2 for 45 min. After incubation, the chambers were washed
twice with 37 °C HBSS to remove any nonadherent neutrophils. W79A
and W79A/R100/101A were diluted to 0.3 µM (final
concentration) in HBSS supplemented with 1.3 mM
CaCl2, and overlaid onto the neutrophils in the appropriate
wells. Neutrophils were incubated with enzyme for 10, 20, 30, and 40 min in a 37 °C, 5% CO2, humidified incubator. At the
specified time, the enzyme was removed, the cells were washed once with
HBSS containing 1.3 mM CaCl2, and then were
fixed at room temperature with 0.4% p-benzoquinone in phosphate-buffered saline (PBS) for 10 min. After fixation, the neutrophils were washed four times with PBS and were placed in blocking
solution (10% normal goat serum and 100 µM goat IgG/ml in PBS) at 4 °C overnight. Then, the monoclonal antibodies to hVPLA2 (26) were diluted to 2 µg/ml in PBS and applied to
the cells. After 1-h incubation at room temperature, the antibodies were removed, and the cells were washed six times in PBS. Secondary antibody, Alexa568 goat anti-mouse (Molecular Probes), was applied for
1/2 h at room temperature. Neutrophils were washed six times with PBS,
and imaged in PBS immediately. Imaging was done with a Zeiss 510 laser
scanning confocal microscope with the detector gain adjusted to
eliminate background autofluorescence contributions.
Expression and Kinetic Characterization of hVPLA2
Mutants--
hVPLA2 is a basic protein (isoelectric
point > 9.0) with eight lysines and seven arginines. A model
structure of hVPLA2 built on the basis of homology to
hIIaPLA2 is illustrated in Fig.
3. The structure suggests that some
cationic residues, including Lys6, Lys11,
Arg34, Lys74, and Arg76, might be
located on its putative interfacial binding surface, whereas others,
including Arg100, Lys101, Lys107,
Arg108, and Arg111, might form a cationic patch
on the opposite face. To determine the roles of these residues in the
interaction of hVPLA2 with cell membranes, we mutated these
cationic residues to glutamate. We also mutated a catalytic residue,
His48, to Ala to generate a catalytically inactive mutant.
Initial attempts to prepare these mutants were hampered by extremely
low refolding efficiency of solubilized inclusion bodies. To overcome this difficulty, we used W79A of hVPLA2, which was shown to
be as active as wild type but much more stable than wild type (18), as
a template for mutant preparation. All these mutants (e.g. K6E/W79A) were expressed in high yields as inclusion bodies and their
refolding yields were uniformly high (i.e. >2 mg/liter of culture after purification).
To determine the effects of mutations on the enzymatic activities of
hVPLA2, we measured the activities of mutants on three types of phospholipid substrates, a short-chain phospholipid
diC6PE, anionic polymerized mixed liposomes, and
zwitterionic [14C]SAPC vesicles. Since the concentration
of diC6PE used in this study (0.5 mM) is well
below the critical micelle concentration for this short-chain
phospholipid (27), diC6PE would exist as a soluble monomer.
Although one cannot preclude the possibility of enzyme-substrate
microaggregate formation (28), the relative activity of mutants
determined under these conditions should thus reflect the relative
catalytic efficiency of their active sites. As summarized in Table
I, a majority of mutants showed
activities comparable to that of W79A (and wild type
hVPLA2), which indicated the intactness of their active
sites. However, three mutants, R34E/W79A, K74E/W79A, and
W79A/K107E/R108E, exhibited less than 35% of wild type activity,
suggesting that these mutations somehow disrupt catalytic steps of
hVPLA2. Since none of these residues are in close proximity
to the active site in the model structure, their deactivating effects
might derive from indirect steric or electrostatic interactions with
the active site residues, as seen with the mutations of other
sPLA2s (10, 29, 30). We then measured the activities of
W79A and mutants on anionic pyrene-PE/BLPG polymerized mixed liposomes
to estimate the effects of mutations on binding to anionic membranes.
If any mutation has a significant effect on binding to anionic
vesicles, it would reduce the activity on pyrene-PE/BLPG polymerized
mixed liposomes to a larger degree than that on diC6PE. As
summarized in the third column of Table I, the relative activity of
mutants on polymerized mixed liposomes was, in general, comparable to
that on the monomeric diC6PE. This indicated that none of
mutated cationic residues is critical for the binding of
hVPLA2 to anionic membranes and the decreased activities of
some mutants derive largely from their reduced catalytic efficiencies. To estimate the effects of mutations on the binding affinity of hVPLA2 for zwitterionic membranes, we then measured the
activities of mutants on SAPC vesicles. We previously showed that
unlike other mammalian sPLA2s, hVPLA2 has
relatively high activity on zwitterionic vesicles and does not show a
significant lag before the initiation of hydrolysis (17, 18). W79A and
mutants also showed linear progress curves up to 10 min with SAPC
vesicles under our assay conditions (data not shown) and, thus, the
specific activities were determined from the slopes of curves after 10 min of incubation. As summarized in the fourth column of Table I, the
relative activity of most of mutants on SAPC vesicles was comparable to
those on other substrates, again indicating that reduced activities of
these mutants derive mainly from their low catalytic efficiencies. A
notable exception was K6E/W79A that had much lower activity on SAPC
vesicles than expected from its activity on diC6PE,
suggesting that the K6E mutation might selectively impair the binding
of enzyme either to zwitterionic membranes or to a PC substrate bound
to the active site. Also, W79A/R100E/K101E showed modestly higher
activity than expected from its activity on other substrates. Finally,
H48A/W79A showed no detectable activities on any phospholipid
substrates even when excess enzyme concentrations were employed
(i.e. >50-fold of W79A concentration).
Membrane Affinities of hVPLA2 and Mutants--
To
quantitatively determine the effects of mutations on the interfacial
binding of hVPLA2, we measured the binding of mutants to
anionic sucrose-loaded BLPG polymerized liposomes and zwitterionic DHPC-coated beads. For PC binding, DHPC-coated beads were used instead
of polymerized liposomes due to low pelleting efficiency of PC
vesicles. Binding constants and relative affinities are summarized in
Table II. The relative affinity for BLPG
polymerized liposomes showed that the mutations of Lys6,
Lys11, and Arg34 of hVPLA2 had
modest effects on its binding to anionic membranes. This supports the
notion that they are located on the interfacial binding surface of
hVPLA2 and accounts for the modest preference of
hVPLA2 for anionic membranes (18). It should be noted that the apparent lack of deactivating effects of these mutations on kinetic
activity toward anionic polymerized mixed liposomes is due to our assay
conditions in which a majority of enzymes are bound to vesicles
(i.e. [BLPG] Heparin Affinities of hVPLA2 and Mutants--
Binding
of hVPLA2 with heparin and its derivatives is a complex
process that involves multiple interaction sites, which makes it
difficult to quantitatively determine the binding affinity (31, 32). We
previously reported the use of immobilized heparin and heparan sulfate
columns to semiquantitatively assess the binding affinities of
hIIaPLA2 and mutants for heparinoids (16). These measurements showed that the relative binding affinities of
hIIaPLA2 and mutants for heparin and heparan sulfate
columns were comparable (16). Thus, we measured the
chromatographic behaviors of hVPLA2 mutants using an
immobilized heparin column to estimate their relative affinities for
cell surface HSPG. The protein bound to the resin was eluted with a
linear gradient of KCl in the elution buffer, and the concentration of
KCl that gives rise to a major protein peak was determined for each
mutant. Chromatograms for selected mutants were shown in Fig.
4, and the KCl concentrations corresponding to protein peaks are listed in Table II. The
chromatographic data clearly showed that the C-terminal cationic
residues, which form a predominant cationic patch on the rear side of
the molecule (see Fig. 3), are involved in heparin binding whereas the
three residues on the front side are not. The presence of a
well-defined heparin-binding site in hVPLA2, which is
spatially distinct from its interfacial binding surface, is in sharp
contrast to a diffuse heparin-binding site of hIIaPLA2
that overlaps considerably with its interfacial binding surface (16).
Despite the presence of well defined heparin-binding site,
hVPLA2 showed significantly lower heparin affinity than did
hIIaPLA2, which eluted with 0.45 ± 0.02 M
KCl. In that hIIaPLA2 has a higher isoelectric point and a
larger number of surface cationic residues than does
hVPLA2, this implies that PLA2-heparin binding
is driven by relatively nonspecific electrostatic interactions.
Activities of hVPLA2 and Mutants to Release Fatty Acids
and Leukotrienes from Neutrophils--
To correlate the different
properties of hVPLA2 mutants to cellular activities, we
measured their activities to release fatty acids from human neutrophils
pre-incubated with radiolabeled AA and OA. With 0.1 µM
W79A, total fatty acid release was 4% and 2% of incorporated AA and
OA, respectively, indicating the lack of pronounced sn-2
acyl selectivity. A similar result was reported for hVPLA2
acting on the macrophage-like cell line, P338D1 (33). Relative activity
of mutants in terms of AA release is listed in Table
III. All mutants showed essentially the
same relative activity in terms of OA release. We also measured their
activities to release LTB4 from human neutrophils, which is
the main eicosanoid product of neutrophils. Results are summarized in
Table III. We previously showed for hVPLA2 and selected
mutants that their activities to release fatty acids from human
neutrophils correlate well with their activities to induce
LTB4 production (18). Within the range of experimental
errors, the mutants listed in Table III showed essentially the same
trend. Note that three mutants, including K6E/W79A, R34E/W79A, and
W79A/K107E/R108E, have significantly lower cell activities than others.
Importantly, all these mutants have a common property, lower
activity on PC vesicles, which derives from different origins:
K6E/W79A has lower PC membrane affinity whereas R34E/W79A and
W79A/K107E/R108E have lower catalytic efficiencies. Among the
three mutants with significantly reduced heparin affinities, only
W79A/K107E/R108E with lower catalytic efficiency showed decreased cell
activities. W79A/R111E with unaltered catalytic efficiencies was as
active as W79A, whereas W79A/R100E/K101E showed even higher activity to
release AA than wild type, which is in accordance with its activity on
SAPC vesicles. Thus, the heparin binding per se does not
appear to play a major role in interaction of hVPLA2 with
neutrophil cell membranes and subsequent leukotriene-inducing activities. Finally, H48A/W79A (up to 1 µM) did not show
detectable activity on neutrophils under our experimental
conditions.
Role of Heparan Sulfate Proteoglycan Binding in hVPLA2
Internalization--
The lack of correlation between the
heparin-binding affinity of hVPLA2 mutants and their
activities on cells, despite relative high affinity of
hVPLA2 for heparin, suggested that the binding to cell
surface HSPG results in a nonproductive process. An interesting possibility is that the HSPG binding of hVPLA2 leads to the
internalization and clearance of surface-bound proteins. The
HSPG-mediated internalization of sPLA2 has been proposed
for group IIa PLA2 (14). To explore this possibility, we
first monitored the temporal and spatial sequences of W79A and
W79A/R100E/K101E exogenously added to neutrophils by
time-dependent immunostaining and confocal microscopic
imaging. Results from the microscopic imaging of the proteins are
illustrated in Fig. 5. The
internalization of W79A to neutrophils was detectable after 10 min
(data not shown) and completed in 20 min. As seen from the comparison
between permeabilized and nonpermeabilized cells, the majority of
W79A molecules were internalized after 20 min. In contrast,
W79A/R100E/K101E showed the same annular distribution on the membrane
whether the cells were permeabilized or not, demonstrating that the
protein molecules are mainly bound to the outer plasma membranes.
Dramatically reduced internalization of W79A/R100E/K101E indicates that
the HSPG binding is important for the internalization of
hVPLA2.
To determine the correlation between the internalization of
hVPLA2 and its eicosanoid-inducing activities, we treated
human neutrophils with the two proteins and measured the time courses of AA and LTB4 release and protein internalization. The
latter was done by time-dependent Western blotting of cell
extracts. Fig. 6 shows that the
liberation of AA by W79A reached a saturated value in about 10 min.
LTB4 release was also completed in 15 min (data not shown).
In contrast, the AA release by W79A/R100E/K101E continued to proceed
even after 1 h, resulting in 75% more AA release than W79A at
1 h (note that data in the second column of Table III were
collected at 30 min). Interestingly, however, the production of
LTB4 by W79A/R100E/K101E ceased in about 15 min (data not
shown). As a result, W79A/R100E/K101E produced higher levels of AA,
but not LTB4, than did wild type (see Table
III). Additionally, cells treated with W79A/R100E/K101E for 1 h
were lysed (data not shown), whereas cells incubated with W79A remained intact under the same conditions. Fig. 7
illustrates the Western blotting of neutrophil extracts treated with
W79A and W79A/R100E/K101E. Since the cells were thoroughly washed with
the buffer containing 0.5 M NaCl, the observed bands should
mainly represent the internalized PLA2. This notion is also
supported by the time-dependent increase in
PLA2 band up to 20 min. The internalized W79A was
subsequently degraded, as indicated by the complete disappearance of
PLA2 after 45 min. In contrast, no internalized
PLA2 band was detected when cells were treated with
W79A/R100E/K101E. Since the production of AA and LTB4 by
W79A was completed before the internalization occurred, it is clear
that the internalization did not result in further production of fatty
acids and LTB4. Also, W79A/R100E/K101E continued to release
AA while acting solely on the outer plasma membranes, again
demonstrating that internalization of hVPLA2 is not
required for the production of fatty acids and LTB4.
Instead, the HSPG-mediated internalization of hVPLA2 might
serve as a mechanism to clear the cell surface-bound hVPLA2
for cell protection.
Mammalian sPLA2s, including group V PLA2,
are mobilized and secreted from various cells in response to
inflammatory stimuli. So far, the mainstay of sPLA2
research has been to study the localization of sPLA2s and
their coupling with cytosolic PLA2 and downstream cyclooxygenases and lipoxygenases during the activation of inflammatory cells. Although these lines of research have produced valuable information about the eicosanoid-inducing actions of
sPLA2s, they did not deliver the detailed mechanistic
information because of inherent difficulty in performing a rigorous
structure-function analysis. We have taken a different approach of
preparing pure recombinant wild type and mutant proteins, thoroughly
characterizing their in vitro enzymatic activities, membrane
affinities, and heparin affinities, and finally measuring their
cellular activities when added exogenously to the cells (16, 18). The
physiological relevance of this approach is supported by findings that
the eicosanoid-inducing actions of exogenously added sPLA2
simulate those of endogenous sPLA2 (12) and that
sPLA2 are released and act both in autocrine and paracrine
manners under inflammatory conditions (7). It should be also stressed
that physiologically relevant submicromolar enzyme concentrations
(i.e. Interfacial Binding and Heparin Binding Residues--
Our model
structure of hVPLA2 (Fig. 3) suggests the presence of
cationic patches on both sides of molecular surface. Membrane and
heparin binding measurements indicate that cationic residues on the
surface containing Trp31, which was shown to be critical
for binding to zwitterionic membranes (18), are involved in binding to
anionic membranes, whereas those on the opposite face are involved in
heparin binding. The effects of the K6E, K11E, and R34E mutations on
the energetics of binding to anionic vesicles, which can be estimated
using an equation,
Our heparin-binding measurements indicate that the five mutated
residues in the carboxyl terminus of hVPLA2, which form a prominent cationic patch in the model structure, provide a binding site
for heparinoids. In case of hIIaPLA2, both amino-terminal and C-terminal cationic patches as well as four cationic residues between residues 53 and 58 were shown to be involved in heparinoid binding (16). Although hVPLA2 also has two cationic
residues, Arg54 and Lys58, on the same surface
as the five C-terminal residues, these residues were not included in
this study because they were shown to be involved in interactions with
the head group of an active site-bound phospholipid (34). The apparent
lack of heparin affinity of Lys6 and Lys11 of
hVPLA2 might be due to their suboptimal arrangement for
heparin binding. The complete spatial separation of interfacial and
heparin binding sites could, in principle, allow the protein molecules bound to cell surface HSPG to act on membrane phospholipids. However, no direct correlation was observed between the in vitro
heparin affinities of mutants and their activities on cells, indicating that the binding of group V PLA2 to cells via HSPG is not
important for its actions on cells. This notion is, however, at odds
with the previous report by Murakami et al. (3), in which
the HSPG affinities of rat group V PLA2 and two mutants,
R94G/K95E and R101S/R102S, which correspond to our K79A/R100E/K101E and
K79A/K107E/R108E mutants (note that different numbering systems are
used; see Fig. 2), are in line with their AA-releasing activities, when
they were transfected into HEK 293 cells. Note, however, that the two measurements were performed using different cell types under different conditions. We performed our studies with neutrophils in the absence of
agonist, whereas Murakami et al. (3) investigated the
augmentation of agonist-induced AA and prostaglandin release by
sPLA2. Although further study is needed to resolve this
controversy, our studies clearly show that K79A/K107E/R108E has low AA-
and LTB4-releasing activities on neutrophils because of its
low catalytic efficiencies.
AA- and LTB4-releasing Activities of
hVPLA2--
Consistent with our previous results on W31A
(18), the relative activity of mutants on PC vesicles are well
correlated with their cell activities. At present, it is not clear how
exactly the K6E mutation reduces the affinity for PC membranes and how the R34E, K74E, and K107E/R108E mutations decreased the catalytic efficiencies. Comparable expression yields of these mutants and W79A
suggest the absence of deleterious gross structural changes that could
result in reduced protein stability. One can thus speculate that the
mutations locally affect the residues involved in interfacial binding,
substrate binding, or catalysis via electrostatic or steric
interactions. Regardless of the origin of these effects, these studies
clearly show that those mutations that reduce the activity of
hVPLA2 on zwitterionic membranes would lower the activity of hVPLA2 to hydrolyze cell membrane phospholipids and
eventually induce eicosanoid formation. This, in conjunction with the
unique ability of hVPLA2 to induce eicosanoid production in
mammalian cells in the absence of agonist (18), indicates that the role of hVPLA2 is to directly bind to the outer plasma membranes
of mammalian cells and to hydrolyze phospholipids, presumably PC. Finally, the lack of cell activities of H48A/W79A points to the absence
of a receptor-mediated mechanism in human neutrophils. Although
H48A/W79A has less than 1% of wild type activity, it can still bind PC
membranes as well as wild type, as shown by the surface plasmon
resonance analysis (data not
shown).2 It also has a
circular dichroism spectrum similar to that of wild type (data not
shown). One would thus expect that it should bind the receptor, if any,
as effectively as the wild type protein. A similar negative result was
observed with mouse group IIa PLA2 acting on HEK 293 cells
(3).
Temporal and Spatial Resolution of hVPLA2
Action--
Our results with doubly labeled neutrophils show that
exogenously added hVPLA2 liberates OA and AA to reasonably
comparable degrees. In view of lack of sn-2 acyl group
specificity of sPLA2s, including hVPLA2, these
findings indicate that exogenously added hVPLA2 is
primarily responsible for the release of fatty acids and
lysophospholipids from neutrophils. For the reasons described above and
below, the site of hVPLA2 action should be the outer plasma
membrane. The elucidation of the exact mechanisms by which these
extracellular fatty acids and lysophospholipids induce cellular eicosanoid production would require further investigation. It is
evident from these studies, however, that the role of
hVPLA2 is not to continuously supply AA for cellular
cyclooxygenases and lipoxygenases from the outer plasma membrane. The
total amount of AA released from the labeled neutrophils is typically
less than 4% of total incorporated AA. The total amount of the
LTB4 release that results from this fatty acid release,
however, is comparable to that induced by potent agonists, such as
N-formyl-methionyl-leucyl-phenylalanine (18). The low AA
release is not because it is mostly converted to LTB4, as
AA and LTB4 release was measured separately under different conditions.
AA release was measured with BSA in the media to ensure that high
proportion of AA liberated from the outer plasma membrane was extracted
into the media, whereas the LTB4 release was measured in
the absence of BSA in the media. It thus appears that the products of
hVPLA2 hydrolysis trigger the cellular eicosanoid
production by activating cellular enzymes involved in eicosanoid
formation. If this is the case, how could cells modulate the extent of
membrane hydrolysis by the cell surface-bound hVPLA2? One
reasonable mechanism is to remove the cell surface-bound hVPLA2 by internalization and degradation. Our confocal
microscopic imaging and Western blotting analysis of neutrophils
treated with hVPLA2 mutants support this notion.
W79A/R100R/K101E with reduced heparin binding affinity was not
internalized into these cells under the conditions where W79A was
internalized and degraded. As a result, the former liberated a larger
amount of fatty acids from the outer plasma membrane than did the
latter under the same conditions. This notion is also consistent with
recent reports that group X PLA2, which has high affinity
for the outer plasma membrane but low affinity for HSPG, is highly
effective in liberating fatty acids from mammalian cells (35, 36).
Since the extensive hydrolysis of the plasma membrane leads to cell
lysis, the overproduction of fatty acids by W79A/R100R/K101E was not
conducive to LTB4 formation, as witnessed by a comparable
degree of LTB4 release by W79A and W79A/R100R/K101E.
The physiological consequence of HSPG-mediated PLA2
internalization has remained controversial. Our results suggest that
HSPG-mediated internalization might serve as a mechanism to remove cell
surface-bound sPLA2 in neutrophils. Apparently, these
results are at odds with a report by Murakami et al. (14),
which indicates that a family of HSPG, glypican, facilitates the
trafficking of group IIa PLA2 into particular subcellular
compartments where it produces AA and induces prostaglandin
biosynthesis. In our studies, no correlation was found between the
internalization of hVPLA2 into neutrophils and the release
of AA and LTB4. W79A completed the AA release before its
internalization took place, whereas W79A/R100R/K101E was more active in
AA release than W79A despite lack of internalization. Again, the
discrepancy might arise from the fact that two studies were performed
using different cells under different conditions. Indeed, Enomoto
et al. (37) recently found that group IIa PLA2 was internalized into mouse bone marrow-derived mast cells and degraded
in a HSPG-dependent manner.
On the basis of our previous and present results, we propose a
mechanism for the action of hVPLA2 on human neutrophils. In this mechanism, exogenous hVPLA2 directly binds the outer
plasma membrane and hydrolyzes PC and other phospholipids to liberate fatty acids and lysophospholipids. This immediate release of
PLA2 products induces the activation of cellular proteins,
including cytosolic PLA2 and 5-lipoxygenase, which
eventually leads to the cellular production of AA and LTB4.
Independently, the HSPG-mediated internalization and degradation of
protein serves as a cell protection mechanism. The presence of distinct
interfacial binding site and HSPG binding site in hVPLA2 is
advantageous for the regulatory purpose because cell surface HSPG can
bind and sequester cell membrane-bound hVPLA2 in action,
without having to compete head-to-head with membrane phospholipids.
Undoubtedly, the detailed understanding of this proposed mechanism
entails further investigation. Additionally, it remains to be seen
whether or not the action of sPLA2s on other mammalian
cells follows the same mechanism. As such, the mechanism provides a
basis for further investigation of the cellular signaling pathways that
lead to the activation of eicosanoid-producing enzymes by exogenous
sPLA2s.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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Fig. 1.
Three hypothetical models of
hVPLA2-cell surface interactions. hVPLA2
can bind either cell surface heparan sulfate proteoglycans that
function as a cell surface adapter for hVPLA2 or a cell
surface receptor specific for hVPLA2. Either binding can
trigger intracellular signaling cascade or lead to internalization of
bound proteins. Heparan sulfate binding can also enhance the apparent
activity of hVPLA2 on membrane phospholipids by increasing
its local concentration. Finally, hVPLA2 can directly bind
the membrane surface and hydrolyze phospholipids. Notice that the
plasma membranes of mammalian cells have asymmetric distribution of
phospholipids, with the outer leaflet rich in sphingomyelin and
phosphatidylcholine.
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Fig. 2.
Partial amino acid sequences of mouse group V
PLA2 (mVPLA2), hIIaPLA2, and
hIIaPLA2. Mutated residues of hVPLA2 are
shown in boldface characters. The numbering
system used is based on the homologous core developed by Renetseder
et al. (38).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. The purity of wild type and mutant proteins,
assessed by sodium dodecyl sulfate (SDS)-polyacrylamide
electrophoresis, was consistently higher than 90%. Protein
concentration was determined by the bicinchoninic acid method (Pierce)
using BSA as standard.
[PL]o, and [E]o are total
phospholipid and enzyme concentrations, respectively. This equation
assumes that each enzyme binds independently to a site on the interface
composed of n phospholipids with dissociation constant of
Kd.
(Eq. 1)
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Fig. 3.
A model structure of hVPLA2 built
on the basis of homology to hIIaPLA2. The model
structure of hVPLA2, shown in space-filling representation,
is built on the backbone of hIIaPLA2 (39, 40) with side
chain replacements using a program, Biopolymer (Molecular Simulation).
The molecule is shown in two orientations: A, the putative
interfacial binding surface; B, the opposite surface facing
the viewer. Cationic side chains are shown in blue, and
mutated ones are labeled. Aliphatic side chains are shown in
yellow, aromatic side chains in green, and
anionic side chains in red. Polar side chains and the
peptide backbone are shown in white.
Relative activities of hVPLA2 mutants versus W79A on
phospholipid substrates
nKd for all
mutants) (29). Mutations of other residues, including Lys74
and Lys76, had much less effect on anionic vesicle binding,
indicating that they are not directly involved in binding to anionic
membranes. When the affinities of mutants for zwitterionic DHPC-coated
beads were measured, none but K6E mutation had a significant effect, indicating that most of anionic residues play no role in binding to
zwitterionic surfaces. The K6E mutation reduced the binding affinity by
about 5-fold, which is much larger than the 2-fold drop in affinity for
anionic BLPG polymerized liposomes. This, in conjunction with the
unusually low kinetic activity of K6E on SAPC vesicles, suggests that
the K6E mutation interferes with the optimal interaction of
hVPLA2 with zwitterionic membranes. However, these results
do not necessarily point to the direct involvement of Lys6
in binding to zwitterionic membranes. Intuitively, it is more likely
that the K6E mutation induces local structural changes of
hVPLA2, which in turn interfere with the interactions of
other critical residues, such as Trp31 (18), with
zwitterionic membranes.
Affinities of hVPLA2 mutants for phospholipids and heparin
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Fig. 4.
Elution of hVPLA2 and mutants
from an immobilized heparin column. Analyzed proteins (40 µg
each) were W79A ( ), W79A/R100E/K101E (
), K79AK107E/R108E (
),
W79A/R111E (
), and hIIaPLA2 (
). Relative activity was
calculated by dividing activity values by an arbitrary value (= 0.03 nmol/min). Note that hIIaPLA2 has significantly lower
activity than hVPLA2 mutants. These data were taken from a
single measurement for each protein.
Activities of hVPLA2 mutants on neutrophils
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Fig. 5.
Internalization of W79A
hVPLA2 and W79A/R100E/K101E. W79A
hVPLA2 (lower panel) and
W79A/R100E/K101E (upper panel) was added to
neutrophils as described under "Experimental Procedures," and the
cells fixed at 20 min after addition of the enzyme. Cells were
incubated with a combination of three different monoclonal antibodies
to hVPLA2 and stained with an AlexaTM 568 goat anti-mouse
secondary antibody (Molecular Probes) with or without permeabilization.
Images were taken on a Zeiss LSM 510 confocal microscope. Top
left, nonpermeabilized cells with W79A/R100E/K101E; top
right, permeabilized cells with W79A/R100E/K101E; bottom
left, nonpermeabilized cells with W79A; bottom
right, permeabilized cells with W79A.
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Fig. 6.
Time course of AA release by W79A
hVPLA2 and W79A/R100E/K101E. 0.1 µM W79A
(open circle) and W79A/R100E/K101E
(closed circle) were incubated with AA-labeled
neutrophils suspended in HBSS plus 0.2% BSA at 37 °C for a given
period. Each data point represents an average of triplicate
measurements.
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Fig. 7.
Western blotting analysis of W79A
hVPLA2 and W79A/R100E/K101E internalized into human
neutrophils. 0.3 µM W79A (A) and
W79A/R100E/K101E (B) were incubated with neutrophils
suspended in HBSS at 37 °C for a given period, and cells were
centrifuged. The pelleted cells were thoroughly washed with HBSS
containing 0.5 M NaCl, lysed, and subjected to
SDS-electrophoresis on 16% acrylamide gels. Essentially the same
electropherograms were obtained from triplicate experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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0.1 µM) were employed in these studies to avoid nonphysiological cell responses. Finally, although one cannot
completely rule out the possibility that the trace amount of bacterial
endotoxin in recombinant hVPLA2 proteins might contribute to their cell activities, the excellent correlation between in vitro and cellular activities of all mutants, no detectable cell activity of H48A/W79A (up to 1 µM) in particular,
indicates that this potential endotoxin effect is insignificant under
our experimental conditions.
G0 =
RT
ln[relative affinity] under the standard conditions with the
concentration of free phospholipid set at 1 M, are 0.54 kcal/mol or less at 25 °C. These values are much smaller than those
calculated for cationic residues on the interfacial binding surfaces of
other sPLA2s, which range from 1.0 to 3.6 kcal/mol (9, 10,
22). Assuming the additivity of their contributions, the total change of interfacial binding energy would be less than 1.5 kcal/mol at
25 °C. This indicates that the membrane binding of
hVPLA2 is driven largely by nonelectrostatic forces. The
fact that hVPLA2 has about 3 times higher affinity for
anionic membranes than hIIaPLA2, despite the much smaller
contributions from its cationic residues, underscores the importance of
the nonelectrostatic interactions. The interactions would derive from a
number of aromatic and aliphatic residues on the putative interfacial
binding surface of hVPLA2 (see Fig. 3). For instance, the
W31A mutation of hVPLA2 alone reduced the interfacial
binding energy by 1.6 kcal/mol (18).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Murakami and Kudo for making their data on mast cells available to us prior to publication.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM52598 (to W. C.) and HL46368 and HL56399 (both to A. R. L.), and by a biomedical science grant from the Arthritis Foundation (to W. C.).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.
¶ Established investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 W. Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho@uic.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M004604200
2 R. V. Stahelin and W. Cho, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BLPG, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol; BSA, bovine serum albumin; diC6PE, 1,2-dihexanoyl-sn-glycero-3-phosphoglycerol; DHPC, 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine; HBSS, Hanks' balanced salt solution; HSPG, heparan sulfate proteoglycan; hIIaPLA2, human group IIa phospholipase A2; hVPLA2, human group V phospholipase A2; LTB4, leukotriene B4; PBS, phosphate-buffered saline; PC, phosphatidyl choline; pyrene-PE, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol; SAPC, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; sPLA2, secretory phospholipase A2; TBS-T, Tris-buffered saline plus 0.05% Tween 20; HEK, human embryonic kidney; OA, oleic acid.
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