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INTRODUCTION |
Phospholipase A2s
(PLA2)1 are a
family of lipolytic enzymes that are found both intra- and
extracellularly. Because PLA2-catalyzed liberation of
arachidonic acid from membrane phospholipids leads to the production of
potent inflammatory lipid mediators, eicosanoids that include
prostaglandins, thromboxanes, leukotrienes, and lipoxins, the
elucidation of their regulatory mechanisms is important for understanding the pathogenesis of inflammatory diseases and for developing a new class of anti-inflammatory drugs. Mammalian tissues contain multiple forms of PLA2s (1), including groups I,
IIa, IIc, V, X (2) secretory PLA2 (sPLA2),
group IV cytosolic PLA2 (cPLA2), and group VI
intracellular Ca2+-independent PLA2. Recent
cell studies have indicated that both cPLA2 and
sPLA2 are involved in eicosanoid production (3-5). The
critical involvement of cPLA2 was demonstrated by recent
genetic studies showing that the disruption of the cPLA2
gene results in loss of lipid mediator biosynthesis (6, 7).
The nature of proinflammatory sPLA2s is not fully
understood. Group IIa sPLA2 has long been implicated in
inflammation based on findings that it is synthesized and secreted by a
variety of cells in response to inflammatory cytokines and that it is
found in fluids from inflammatory exudation. However, group IIa
sPLA2 has extremely low affinity for zwitterionic
phosphatidylcholine (PC) vesicles (8, 9). Thus, it is unclear how this
secreted protein might act on the extracellular face of plasma membrane of mammalian cells, which is composed largely of zwitterionic phospholipids, PC and sphingomyelin. More recently, group V
sPLA2 has been shown to be involved in eicosanoid formation
from murine macrophages and mast cells (10, 11). Molecular cloning of group V sPLA2 from different species showed that these
enzymes, although homologous to group IIa sPLA2, have some
notable variations in amino acid sequence (12, 13). In particular, they
contain a few tryptophan residues, some of which are located on their putative interfacial binding surfaces (see Fig. 1), whereas group IIa
sPLA2s have none. Those sPLA2s (e.g.
cobra venom PLA2s) that show high activity toward PC
vesicles and intact cell membranes typically have a number of
tryptophans and other aromatic side chains on their interfacial binding
surfaces; this finding suggests that group V sPLA2 might be
better suited for acting on the outer cell membrane than group IIa
sPLA2. Indeed, our recent study demonstrated that human
group V sPLA2 (hsPLA2-V) could bind and
hydrolyze PC vesicles much more effectively than human group IIa
sPLA2 (hsPLA2-IIa) (14). The present study
demonstrates that hsPLA2-V also is much more active than
hsPLA2-IIa in releasing fatty acids (including arachidonic
acid) and in eliciting eicosanoid production from a variety of cells
including neutrophils. This study also identifies a surface tryptophan
residue (Trp31) of hsPLA2-V as a structural
determinant of its high affinity for PC membranes and for outer
cell membranes.
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EXPERIMENTAL PROCEDURES |
Materials--
1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine
(pyrene-PC), and -glycerol (pyrene-PG) were purchased from Molecular Probes (Eugene, Oregon).
1,2-Bis(12-(lipoyloxy)-dodecanoyl)-sn-glycero-3-phosphoglycerol (BLPG) was prepared as described elsewhere (15, 16).
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
and -glycerol (POPG),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and
-methanol (DMPM), and
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine were
from Avanti Polar Lipids. Polymyxin B sulfate, Naja naja naja venom PLA2,
1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC), 1,2-sn-dioleoylglycerol, cytochalasin B, and
N-formylmethionyl-leucyl-phenylalanine were from Sigma.
Racemic
1,2-dihexanoylthio-1,2-dideoxy-glycero-3-phosphosphocholine (diC6thio-PC) was prepared as described (17).
Phospholipid concentrations were determined by phosphate analysis (18).
1-Stearoyl-2-[14C] arachidonoyl-sn-glycero-3-phosphocholine
(55 mCi/mmol) was 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 was from Bayer Inc. (Kankakee, Illinois).
5,5'-Dithiobis(2-nitrobenzoic acid) and sodium sulfite were obtained
from Aldrich. 2-Nitro-5-(sulfothio)-benzoate was synthesized from
5,5'-dithiobis(2-nitrobenzoic acid) as described (19). All restriction
enzymes, T4 ligase, T4 polynucleotide, kinase and isopropyl
-D-thiogalactopyranoside were obtained from Boehringer
Mannheim. Oligonucleotides were purchased from Integrated DNA
Technologies (Coralville, IA) and used without further purification. Recombinant hsPLA2-IIa, which carries the Asn1
to Ala mutation, was prepared as described (20-22). Porcine pancreatic PLA2 was obtained as a gift from Prof. M. K. Jain
(University of Delaware).
Mutagenesis--
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 (23).
Expression and Purification of
hsPLA2-V--
Proteins were expressed in Escherichia
coli, refolded, and purified as described previously (14), with
some modifications. E. coli strain BL21(DE3) was used as a
host for protein expression. An 8-liter Luria broth containing 100 µg/ml of ampicillin was inoculated with 80 ml of overnight culture
from a single colony and was grown at 37 °C. When the absorbance of
the medium at 600 nm reached 0.2, additional ampicillin was added to a
final concentration of 1 mM, and 0.5 mM
isopropyl
-D-thiogalactopyranoside was added when the
absorbance at 600 nm reached 0.8. After an additional 4 h at
37 °C, cells were harvested at 3000 × g for 10 min
at 4 °C and frozen at -20 °C. Cells were resuspended in 100 ml
of 0.1 M Tris-HCl buffer, pH 8.0, containing 5 mM EDTA, 50 mM NaCl, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5% (v/v) Triton X-100, and 0.4%
(w/v) sodium deoxycholate and stirred at 4 °C. The suspension was
sonicated on ice using a Sonifier 450 (Branson) in pulse mode for 10 pulses of 15 s each. The inclusion body pellet was obtained by
centrifugation at 17,000 × g for 20 min. The pellet
was resuspended in 0.1 M Tris-HCl buffer, pH 8.0, containing 5 mM EDTA, 50 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.8% (v/v) Triton X-100,
and 0.8% (w/v) sodium deoxycholate, sonicated as described above, and
the suspension was centrifuged. The pellet was resuspended in 100 ml of
the same buffer solution and stirred for 30 min at room temperature.
The pellet was collected by centrifugation as described above and
washed in 50 ml of 50 mM Tris-HCl, pH 8.0, containing 5 M urea and 5 mM EDTA, and the suspension was
centrifuged. The inclusion body protein was solubilized in 20 ml of 50 mM Tris-HCl, pH 8.5, containing 8 M guanidinium
chloride and 0.3 M sodium sulfite, and stirred vigorously
at room temperature for 30 min. Eight ml of
2-nitro-5-(sulfothio)-benzoate solution (50 mM) was then
added, and the modification was monitored spectrophotometrically at 412 nm. After the modification was complete (approximately 20 min), the
mixture was further stirred for 20 min, and any insoluble matter was
removed by centrifugation at 100,000 × g for 15 min at
room temperature. The reaction mixture was loaded onto a Sephadex G-25
column (2.5 × 45 cm) equilibrated with 25 mM Tris-HCl
buffer, pH 8.0, containing 5 M urea and 5 mM
EDTA at room temperature, and the second major protein peak was
collected (120 ml) and dialyzed against water and then against 0.3%
(v/v) glacial acetic acid to precipitate the sulfonated protein. The
precipitated protein was resuspended in 100 ml of deionized water, and
the suspension was centrifuged at 17,000 × g for 10 min at
4 °C. The protein pellet was resuspended in 10 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM
EDTA and 5 M guanidinium chloride. The clear solution was
loaded onto a HiLoad 16/60 Superdex 200 column (Amersham Pharmacia Biotech) attached to a fast protein liquid chromatography system at
4 °C (Amersham Pharmacia Biotech). The major protein peak was collected (ca 30 ml), and to this solution of sulfonated protein, 30 ml
of 50 mM Tris-HCl, pH 8.5, containing 10% (v/v) glycerol, 8 mM reduced glutathione and 7 mM oxidized
glutathione, were added dropwise with stirring (120 rpm) over 3 h.
The solution was kept at room temperature for 20 h, at which
point, the protein solution was dialyzed at room temperature against 3 volumes of 4 liters of 25 mM Tris-HCl buffer containing 0.2 M guanidinium chloride and 10% (v/v) glycerol, pH 7.5. For
wild type hsPLA2-V, the clear solution of folded protein
was fractionally precipitated with 30-40% ammonium sulfate at room
temperature. The resulting protein precipitate was collected by
centrifugation at 50,000 × g for 15 min at 4 °C and
resuspended in 5 ml of 25 mM Tris (pH 7.5) buffer
containing 0.2 M guanidinium chloride. The fractional
precipitation of refolded hsPLA2-V by ammonium sulfate not
only increased the purity of protein (>90% pure electrophoretically)
but also allowed the concentration of protein solution (up to
micromolar). For W31A and W78A, the refolded protein solution was
dialyzed at room temperature against 25 mM Tris-HCl buffer,
pH 7.5, containing 0.2 M guanidinium chloride, then against
25 mM Tris-HCl, pH 7.5, and finally against distilled
water. The dialyzed solution was lyophilized and the lyophilized powder
was stored at -20 °C. The purity of wild type and mutant proteins
assessed by SDS-polyacrylamide gel electrophoresis was consistently
higher than 90%. Protein concentration was determined by the
bicinchoninic acid method using bovine serum albumin as standard (Pierce).
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-containing phospholipids (1 mol %) inserted in 9.9 µM BLPG, 2 µM bovine serum albumin, 0.16 M NaCl, and 10 mM CaCl2 (15, 16).
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 (22). Enzymatic
hydrolysis of DMPC and DMPM vesicles was monitored with the pH-stat
method (24). Sonicated small unilamellar vesicles of DMPM
were prepared as described (24). Sonicated small
unilamellar DMPC vesicles were prepared by suspending 10 mg of lipid in
1 ml of water and sonicating as described (for about 10 min to give an
almost clear suspension). The vesicles were annealed by incubating the
solution at 50 °C for 90 min and then kept at 37 °C during use
over 1 day. DMPM reaction mixtures contained 4 ml of 1 mM
NaCl, 2.5 mM CaCl2, 20 µg of polymyxin B
sulfate, 240 µM DMPM at 21 °C and pH 8.0. DMPC reaction mixtures contained 100 mM NaCl, 1 mM
CaCl2, 77 mM DMPC in a volume of 4 ml at
25 °C and pH 8.0. Reactions were started by the addition of enzyme
and monitored as the consumption of 3 mM NaOH titrant.
Assays were calibrated to give nmol of product as described (24).
Assays with monomeric substrate racemic diC6thio-PC were
carried out as described (17).
Binding of sPLA2 to Phospholipid-coated Beads and
Vesicles--
To circumvent the complication due to low pelleting
efficiency of PC vesicles, PC-coated styrene-divinylbenzene beads that can be rapidly and completely separated from the solution by low speed
centrifugation were used (9). Phospholipid-coated beads were prepared
as described previously (9). Phospholipid-coated beads were suspended
in 3 ml of 10 mM Tris-HCl buffer, pH 7.4, containing 0.16 M NaCl and 0.1 mM EDTA (or 10 mM
CaCl2; see under "Results"). Final bulk phospholipid
concentration was 100-150 µM. Aliquots (20-140 µl) of
bead suspension was incubated at room temperature for 15 min in the
same buffer (total volume, 150 µl) containing 1 µM of
bovine serum albumin and varying concentrations of PLA2.
Controls contained the same mixtures minus phospholipid-coated beads.
Mixtures were centrifuged for 2 min at 12,000 × g, and aliquots of the supernatants were assayed for PLA2 activity
using 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine/POPG/1,2-sn-dioleoylglycerol (10:9:1 in mol ratio) mixture as a substrate as described (25). Values
of n and Kd were determined by nonlinear
least-squares analysis of the [E]b
versus [E]o plot using the following
equation,
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(Eq. 1)
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where [PL]o,
[E]o, and [E]b are total
phospholipid, total enzyme, and bound enzyme concentrations, respectively. This equation assumes that each enzyme binds
independently to a site on the interface composed of n
phospholipids with a dissociation constant of Kd.
For anionic PG, the binding was measured in the presence of 1 mM EDTA using both POPG-coated beads and sucrose-loaded
POPG vesicles. The binding to sucrose-loaded POPG vesicles was measured
as described previously (22).
Monolayer Experiments--
Surface pressure (
) of monolayers
was measured at room temperature using a du Nouy ring as described
previously (26, 27). DHPC was spread onto the subphase (20 mM Tris-HCl, pH 7.5, 0.16 M NaCl, and 10 mM Ca2+) to form a monolayer with a given
initial surface pressure (
o). Then, PLA2 was
injected into the subphase and penetration was measured by monitoring
the change in surface pressure (
). At a given
o of
phospholipid monolayer, the maximal
value depended on the
protein concentration in the subphase and reached a saturation when the
protein concentration was above a certain value (approximately 1.5 µg/ml of hsPLA2-V at
o = 5 dyn/cm). Protein
concentrations in the subphase were therefore maintained above such
values to ensure that an observed 
value represents a maximum at
a given
o. The analysis of monolayer penetration data
obtained under this condition was described in detail previously
(25).
Fatty Acid Release from Mammalian Cells--
Fatty acid release
from CHO-K1 and RAW 264.7 cells and from human peripheral blood
neutrophils by exogenously added PLA2s was measured using a
real-time fluorometric assay based on rat liver fatty acid-binding
protein as described (28).
Eicosanoid Production from Neutrophils--
Human neutrophils
were prepared from heparinized venous blood collected from healthy
medication-free donors by fractionation through centrifugation on
Percoll solution for 20 min at 1000 × g, and remaining
red blood cells were removed by hypotonic lysis as described previously
(29). Neutrophils (1 × 106 cells/ml) were incubated
at 37 °C in 250 µl of Hanks' balanced salt solution containing
CaCl2 (1.2 mM) and increasing concentrations (1-100 nM) of hsPLA2-V, W31A, W79A, or
hsPLA2-IIa. Control cells were treated with Hanks'
balanced salt solution. Thereafter cells were centrifuged at 8000 × g for 2 min. Leukotriene levels were determined using a
leukotriene B4 (LTB4) enzyme immunoassay kit from Cayman Chemical Co. (Ann Arbor, MI). Typically, LTB4
secretion reached a maximal value within 30 min of incubation under
this condition. The maximal value for each incubation mixture was then corrected for a background signal from control cells.
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RESULTS |
Kinetic Activities of hsPLA2-V and
Mutants--
hsPLA2-V has four tryptophan residues, of
which at least two are solvent-exposed according to the model structure
of hsPLA2-V, based on homology between it and
hsPLA2-IIa (Fig. 1). To
assess the contribution of surface tryptophan residues of
hsPLA2-V to its unique ability to act on PC membranes, two
surface tryptophans, Trp31 and Trp79, were
mutated to Ala. Both mutants, W31A and W78A, were refolded more
effectively than wild type, suggesting that these surface tryptophans
might interfere with the in vitro refolding of recombinant hsPLA2-V. Enzymatic activities of wild type
hsPLA2-V and the mutants were then rigorously compared
using different types of substrates: monomers, anionic and zwitterionic
vesicles, and polymerized mixed liposomes. We first measured initial
velocities for hsPLA2-V-catalyzed hydrolysis of the soluble
substrate racemic diC6thio-PC (see Table I). The substrate concentration, 0.5 mM, is well below the critical micelle concentration for
this short-chain phospholipid (17). The turnover numbers for
hsPLA2-V and its two mutants are comparable to that
measured with N. n. naja PLA2, suggesting that
the recombinant hsPLA2-V and mutants are correctly
refolded. It is not clear whether hydrolysis of this substrate occurs
via a truly monomeric enzyme-substrate complex or whether enzyme and
substrate interact to form a enzyme-substrate microaggregate (30).

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Fig. 1.
A model structure of hsPLA2-V
based on homology to hsPLA2-IIa. The model structure
of hsPLA2-V shown as a space filling representation is
built on the backbone of hsPLA2-IIa (42, 43) with side
chain replacements using the Biopolymer program (Molecular
Simulations). The molecules are oriented with their (putative)
interfacial binding surfaces facing the viewer. Two mutated surface
tryptophans of hsPLA2-V are shown in red and
labeled. Aliphatic side chains are shown in yellow, aromatic
side chains in green, cationic side chains in
blue, and anionic side chains in pink. Polar side
chains and the peptide backbone are shown in white.
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Table I
Rate constants for in vitro hydrolysis of substrates by PLA2s
Turnover numbers (kcat) for diC6thio-PC,
DMPC, and DMPM were determined from initial velocities of hydrolysis as
described under "Experimental Procedures." Specificity constants
(kcat*/Km*) for polymerized mixed
liposomes were determined from the nonlinear least-squares analysis of
reaction progress curves.
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The turnover numbers calculated from the initial velocities for the
hydrolysis of anionic DMPM vesicles by hsPLA2-V, W31A and
W79A, and hsPLA2-IIa are given in Table I. In these assays, the cationic cyclic peptide polymyxin B was included. This additive causes rapid intervesicle exchange of DMPM, which keeps the mole fraction of nonhydrolyzed DMPM in enzyme-containing vesicles near 1 so
that the initial velocity can be easily measured (24). The progress
curves were linear for at least 15 min (not shown). The turnover number
under these conditions for hsPLA2-V is 13-fold smaller than
that for hsPLA2-IIa, and the mutation of Trp31
and Trp79 to Ala has only a modest effect on the catalytic
efficiency. When the concentration of DMPM was doubled from 240 to 480 µM, the initial velocities changed by <5%, which proves
that all of the proteins are fully bound to vesicles. Thus, the
differences in the turnover numbers reported in Table I reflect
differences in the catalytic efficiencies of the enzymes at the
vesicle interface.
Fig. 2 shows the reaction progress curves
for the hydrolysis of DMPC vesicles by PLA2s, and Table I
gives the turnover numbers. As expected from earlier studies,
hsPLA2-IIa showed very poor activity on zwitterionic
vesicles, and this was due in part to poor binding to vesicles that
lack negative charge (8, 21). The progress curve with porcine
pancreatic PLA2 shows the classical lag phase, which is due
in part to poor binding of enzyme to non-hydrolyzed vesicles and
product-dependent binding of enzyme leading to rate acceleration (31). Even in the presence of 13.5 µg of porcine pancreatic PLA2, the initial rate was barely detectable,
and then the velocity accelerated dramatically after about 10 min (Fig. 2). Interestingly, wild type and W79A hsPLA2-V were highly
active on DMPC vesicles, and only a very short lag was seen (Fig. 2). The turnover numbers for both proteins measured after the short lag
were approximately 7000-fold larger than that for
hsPLA2-IIa (Table I). This is in marked contrast to the
results with DMPM vesicles, in which hsPLA2-IIa is the more
active enzyme. Mutation of Trp31 to Ala reduced the
turnover number by 44-fold. Thus, Trp31 seems to be a key
residue for promoting high activity of hsPLA2-V on
zwitterionic vesicles.

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Fig. 2.
Reaction progress curves for the hydrolysis
of DMPC vesicles by PLA2s. Curve a, 2 µg of W79A hsPLA2-V; curve b, 2 µg of wild
type hsPLA2-V; curve c, 13.5 µg of porcine
pancreatic PLA2; curve d, 2 µg of W31A
hsPLA2-V; curve e, 60 µg of
hsPLA2-IIa. Reaction conditions are given under
"Experimental Procedures."
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We also measured the activity of hsPLA2-V and mutants on
anionic polymerized mixed liposomes. In polymerized mixed liposome system, it is possible to accurately determine the head group specificity of PLA2 by varying the head group structure of
hydrolyzable phospholipids in an inert polymerized matrix. Two
phospholipids, pyrene-PC and pyrene-PG, were used as inserts in the
BLPG polymerized matrix. As reported previously (14),
hsPLA2-V has comparable activities on pyrene-PC and
pyrene-PG, whereas hsPLA2-IIa has much lower activity on
pyrene-PC. As a result, hsPLA2-V is about 35 times more
active than hsPLA2-IIa on pyrene-PC/BLPG polymerized mixed
liposomes and 5 times less active than hsPLA2-IIa on
pyrene-PG/BLPG polymerized mixed liposomes. Most importantly, the
effects of W31A and W79A mutations on the activities of
hsPLA2-V on the two polymerized mixed liposomes are
comparable, indicating that the mutations have no effect on the head
group specificity of hsPLA2-V. Thus, the effects of W31A
mutation on kinetic activities of hsPLA2-V are solely due
to the reduced interfacial binding. All together, the data in Table I
indicate that the dramatically lower activity of hsPLA2-IIa
on PC versus anionic PG interfaces is due to a combination of its poor interfacial binding to zwitterionic interfaces and lower
preference of its catalytic site for PC. Also, the data indicate that
hsPLA2-V has much greater activity on PC than does hsPLA2-IIa because of its higher affinity for zwitterionic
interfaces and comparable affinity of its active site for PC and
anionic phospholipids.
Membrane Affinities of hsPLA2-V and Mutants--
To
further study how Trp31 promotes the high activity of
hsPLA2-V on zwitterionic PC vesicles, we measured the
binding affinity of wild type and mutants for PC- and PG-coated beads.
Phospholipid-coated hydrophobic beads have been shown to be useful in
determining the membrane affinity of PLA2s (9). In
particular, this model membrane allows rapid and accurate measurement
of PC affinity, which normally is difficult to achieve with PC vesicles
due to their low pelleting efficiency compared with anionic vesicles (data not shown).
We first measured the binding affinity of hsPLA2-V for
beads coated with DHPC, a nonhydrolyzable ether analog of PC. Note that
Kd is expressed in terms of molarity of enzyme
binding sites composed of n phospholipids (Equation 1).
Thus, nKd is the dissociation constant in terms of
molarity of lipid molecules and the relative binding affinity can be
best described in terms of relative values of
(1/nKd). As shown in Fig.
3, hsPLA2-V had relatively
high affinity for DHPC-coated beads in the presence and absence of
Ca2+; nKd = 1.5 ± 0.3 µM with 10 mM Ca2+ and = 1.8 ± 0.3 µM with 0.1 mM EDTA. Under
the same conditions, hsPLA2-IIa showed much lower affinity
(i.e. nKd > 100 µM).

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Fig. 3.
Binding isotherms of hsPLA2-V and
hsPLA2-IIa. The binding of hsPLA2-V to
DHPC-coated beads (10 µM of bulk phospholipid
concentration) was measured in the presence of 10 mM
Ca2+ ( ) or 0.1 mM EDTA ( ). The binding of
hsPLA2-V ( ) and hsPLA2-IIa ( ) to
POPC-coated beads (10 µM) was measured in the presence of
0.1 mM EDTA. Each point represents an average of duplicate
measurements. Solid lines are theoretical curves constructed
using Equation 1 with experimentally determined n and
Kd values.
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The Ca2+ independence of the interfacial binding of
hsPLA2-V allowed us to measure the binding to PC- and
PG-coated beads in the absence of Ca2+ using natural
phospholipids instead of ether analogs. hsPLA2-V showed
essentially the same affinity for DHPC and POPC-coated beads in the
absence of Ca2+ (see Fig. 3). Thus, we measured the
relative affinity of wild type hsPLA2-V and mutants for
beads coated with readily available POPC and POPG in the absence of
Ca2+ (i.e. with 0.1 mM EDTA).
nKd and relative affinity values are summarized in
Table II. The relative affinity is
calculated as the ratio of 1/nKd value for
hsPLA2-V/POPC-coated beads binding to that for other
enzyme/lipid combinations. Compared with wild type
hsPLA2-V, W31A mutant bound 14 times less tightly to the
PC-coated beads, whereas W79A retained about one-half of the wild type
affinity. This indicates that Trp31 plays an important role
in the binding of hsPLA2-V to PC membranes, whereas
Trp79 is not directly involved in the process.
hsPLA2-V showed about 15-fold higher binding for
POPG-coated beads than for POPC-coated beads. This is much smaller than
the >200-fold increase in binding observed for hsPLA2-IIa.
hsPLA2-V had 2-fold higher affinity for PG membranes than
did hsPLA2-IIa. Thus, hsPLA2-V has high
intrinsic affinity for both zwitterionic and anionic membranes, whereas hsPLA2-IIa has high affinity only for anionic interfaces.
Unlike the case with PC-coated beads, the effect of the
Trp31 to Ala mutation on the affinity of
hsPLA2-V for PG-coated beads was not pronounced
(approximately a 2.3-fold drop) and comparable to the effect of
the Trp79 to Ala mutation (3.8-fold decrease). This
indicates that Trp31 plays a less critical role in the
binding of hsPLA2-V to anionic surfaces, which may be
driven in part by electrostatic interactions.
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Table II
Membrane binding affinities of wild type and mutant hsPLA2-V
and hsPLA2-IIa
Values represent the mean and S.D. of triplicate determinations.
Relative affinity is the ratio of 1/nKd value of
hsPLA2-V for POPC-coated beads to that for other enzyme/lipid
combinations.
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Finally, we measured the binding of enzymes to sucrose-loaded POPG to
check the validity of our binding data using phospholipid-coated beads.
As shown in Table II, all PLA2s showed 3-4-fold higher affinity for POPG vesicles than for POPG-coated beads, which presumably reflects different surface packing density and curvature of the two
model membranes. Importantly, the relative affinity of
PLA2s for POPG vesicles was essentially the same as that
for POPG-coated beads, demonstrating the validity of our uses of
phospholipid-coated beads to quantify the affinity of PLA2s
to biological membranes.
Monolayer Penetration of hsPLA2-V and Mutants--
To
better understand how Trp31 contributes to interfacial
binding of hsPLA2-V, we measured the penetration of wild
type hsPLA2-V and mutants into the DHPC monolayer at the
air-water interface. In these studies, a phospholipid monolayer of a
given initial surface pressure (
o) was spread at constant
area and the change in surface pressure (
) was monitored after
injection of protein into the subphase. Fig.
4 shows that W31A penetrates into DHPC
monolayer significantly less effectively than wild type and W79A over a
wide range of
o. The monolayer penetration ability of W31A
was similar to that of hsPLA2-IIa, which has extremely low
activity on PC monolayers and bilayers. This data thus suggests that
the higher activity of hsPLA2-V on PC membranes derives
from the ability of Trp31 to partially penetrate into
zwitterionic PC membranes, thereby making favorable interfacial
interactions.

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Fig. 4.
Penetration of hsPLA2-V ( ),
W31A ( ), W79A ( ) and hsPLA2-IIa ( ) into the DHPC
monolayer as a function of initial surface pressure of monolayer.
The subphase contained 20 mM Tris-HCl, pH 7.5, 0.16 M NaCl, and 10 mM Ca2+. Enzyme
concentrations were 0.12 µM. Solid lines were
obtained by the linear regression of experimental data. Each point
represents an average of duplicate measurements
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Activities of hsPLA2-V and Mutants to Release Fatty
Acids and Eicosanoids from Cells--
The extracellular face of the
plasma membrane of mammalian cells is largely composed of zwitterionic
PC and sphingomyelin. Thus hsPLA2-V, which has higher
affinity and activity for PC membranes than does
hsPLA2-IIa, might show relatively high activity on the outer cell membrane. The activities of hsPLA2-V and mutants
as well as hsPLA2-IIa, added exogenously to the mammalian
cell lines including RAW264.7 and CHO-K1, were measured by monitoring
fatty acid release. Also, N. n. naja PLA2 which
is highly active on PC vesicles and intact cells was studied for
comparison. As summarized in Table III,
hsPLA2-V was 20-30 times more active than
hsPLA2-IIa but 15-20-fold less active than N. n.
naja PLA2 on these cells. Also, W31A
hsPLA2-V had 10-30% of the wild type activity, and W79A
showed only a modest decrease in activity. Thus, both the difference in
activity between hsPLA2-IIa and hsPLA2-V and
the effects of tryptophan mutations on the activity of
hsPLA2-V were less pronounced in cell assays than in
vesicle assays. It should be noted, however, that there are differences
between vesicles and complex cell membranes, including the fact that
the latter has some outer layer anionic lipids. Thus, the qualitative
correlation between the activities of wild type and mutants determined
from cell and vesicle assays supports the notion that the high activity of hsPLA2-V to release fatty acids from mammalian cells
derives from its ability to avidly bind PC membranes and that
Trp31 plays an important role.
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Table III
Cellular activities of hsPLA2-V, its tryptophan mutants, and
hsPLA2-IIa
Values represent the mean of triplicate determinations.
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We also measured the release of fatty acids and LTB4 from
human neutrophils by various exogenously added sPLA2s,
including hsPLA2-V and mutants, to see whether their
activity to release fatty acids from the outer cell membrane is
correlated to their ability to elicit cellular eicosanoid production.
LTB4 is a major eicosanoid produced by neutrophils upon
activation by inflammatory agonists. All sPLA2s showed
comparable activities on neutrophils, RAW264.7, and CHO-K1 cells. As
shown in Fig. 5, hsPLA2-V
elicited LTB4 production in a
concentration-dependent manner from unstimulated human
neutrophils. In contrast, hsPLA2-IIa showed less than 10% of the hsPLA2-V activity under the same conditions.
hsPLA2-V (100 nM) released 190 pg of
LTB4 per 106 cells, which is half of the
maximal amount of LTB4 release (400 pg/106
cells) caused by the potent activators, f-MLP (1 µM) + cytochalasin B (5 µg/ml), under the same conditions. At the same
concentration, W79A and W31A showed 77 and 29% of the wild type
activity, respectively. Overall, an excellent correlation was observed
between the relative activity of sPLA2s to release of fatty
acids and that to elicit LTB4 production from neutrophils.
Taken together, these results indicate that hsPLA2-V has
higher activities to release fatty acids, including arachidonic acid,
from the outer cell membrane and to elicit cellular eicosanoid
production than does hsPLA2-IIa and that Trp31
plays an important role in these activities.

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Fig. 5.
Dose-dependent LTB4
release from human neutrophils by exogenous
hsPLA2-V ( ), W31A ( ), W79A ( ), and
hsPLA2-IIa ( ). Incubation mixtures at 37 °C
contain neutrophils (0.5 × 106 cells) and varying
concentration of enzymes in Hanks' balanced salt solution.
LTB4 levels were determined after 30 min incubation. Each
point represents an average of quadruple measurements.
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DISCUSSION |
This study demonstrates that hsPLA2-V is more active
than hsPLA2-IIa by up to 4 orders of magnitude in
hydrolyzing PC-rich membranes, including the outer plasma membrane of
mammalian cells. sPLA2s have a common interfacial binding
surface that is located on the flat external surface surrounding the
active site slot. Many sPLA2s, including
hsPLA2-IIa, prefer anionic membranes due in part to the
presence of cationic residues on the interfacial binding surface. Only
a subset of sPLA2s (e.g. cobra
PLA2s) that contain a number of aromatic residues, Trp in
particular, on their interfacial binding surfaces can effectively bind
and hydrolyze PC membranes. For instance, N. n. naja
PLA2 has three tryptophans on its putative interfacial
binding surface, and this may be the reason it shows the highest
activity and affinity for PC membrane and intact cells (32). Also,
mutational analyses of several sPLA2s demonstrated the
importance of surface tryptophans in interfacial binding (33-35). In
particular, the addition of a single tryptophan to the interfacial
binding surface of hsPLA2-IIa enhances its activity on PC
membranes by more than 2 orders of magnitude (35). Mammalian group V
PLA2s contain multiple tryptophans (three for mouse and rat
enzymes and four for hsPLA2-V) among which
Trp31 and Trp43 are conserved (13). The model
structure of hsPLA2-V illustrated in Fig. 1 shows that
Trp31 is located in the center of its putative interfacial
binding surface, thereby suggesting its critical involvement in
interfacial binding. This study shows that Trp31 indeed
plays an essential role in the binding of hsPLA2-V to membranes, zwitterionic PC membranes in particular, whereas
Trp79 located on the opposite face is involved neither in
interfacial binding nor in catalytic steps. Reduced enzymatic activity,
vesicle binding affinity and monolayer penetration power of W31A
compared with wild type enzyme show that Trp31 enhances the
binding of hsPLA2-V to membranes, whether zwitterionic or
anionic, by partially penetrating into membranes and thereby achieving
optimal interactions with membranes, which involve a complex
combination of hydrophobic and electrostatic interactions (36).
hsPLA2-V also contains several cationic residues in its
putative interfacial binding surface (see Fig. 1). The fact that
hsPLA2-V prefers anionic PG membranes to PC membranes
suggests the importance of these cationic residues in its membrane
binding and possibly cell surface binding (5). However, other features
in addition to electrostatics are involved in promoting the relatively
high affinity of sPLA2s for anionic versus
zwitterionic membranes (37). Higher affinity of hsPLA2-V
for anionic membranes than hsPLA2-IIa despite the smaller
number of cationic interfacial binding residues again underscores the
contribution of nonelectrostatic effects. Presumably, Trp31
and other non-ionic residues also make a significant contribution to
the binding of hsPLA2-V to anionic membranes.
As shown in Fig. 2, the hydrolysis of PC membranes by porcine
pancreatic PLA2 is preceded by a long lag, which
corresponds to an accumulation of a critical amount of reaction
products, one of which is anionic fatty acid. Jain and Berg (31) have shown that addition of PLA2 reaction products to PC
vesicles greatly promotes the binding of the porcine enzyme to the
interfaces, and this binding enhancement provides a basis for the rate
acceleration. Cobra venom PLA2s bind tightly to PC
vesicles, and no lag in the hydrolysis of PC vesicle is seen (31).
hsPLA2-V binds less tightly to PC membranes than do cobra
venom PLA2s but much more tightly than do
hsPLA2-IIa and porcine pancreatic PLA2, which
is consistent with a short lag seen in the hydrolysis of PC vesicles by
hsPLA2-V. The unique ability of hsPLA2-V to
avidly bind both zwitterionic and anionic membranes would not only
shorten the lag for the initial hydrolysis of PC membranes but also
allow the enzyme to remain on membrane surfaces as the hydrolysis
progresses with the accumulation of anionic fatty acids. This might
account for the larger effect of the W31A mutation on the kinetics of
DMPC hydrolysis (44-fold) than on the binding to PC membranes
(15-fold). Furthermore, hsPLA2-V has an additional kinetic
advantage over hsPLA2-IIa in that the catalytic site of the
former can accommodate both zwitterionic and anionic phospholipids
whereas the catalytic site of the latter discriminates against PC
(Table I). Note that the catalytic activity of hsPLA2-V is
approximately 13 times and 5 times lower than that of
hsPLA2-IIa toward DMPM vesicles and pyrene-PG/BLPG
polymerized mixed liposomes, respectively, under conditions where all
enzymes are bound to vesicles. This difference in intrinsic catalytic activity on anionic phospholipids is presumably due to their different active site structures. Our previous mutagenesis study of
hsPLA2-IIa showed that the mutation of its
Lys69, which forms a hydrogen bond with the sn-3
phosphate of substrate, to Arg resulted in a 5-fold reduction in
catalytic activity toward anionic phospholipids (21). Sequence
comparison of hsPLA2-IIa and hsPLA2-V reveals
that the latter has Arg in place of Lys in position 69 (13). The origin
of unique substrate head group specificity of hsPLA2-V is
currently under investigation.
Our data show good correlation between the in vitro ability
of sPLA2s to act on model membranes, such as vesicles, and
their activity on complex cell membranes. Such a correlation has also been observed for the action of a panel of hsPLA2-IIa
mutants on vesicles and cell membranes (28). Exogenously added
hsPLA2-IIa, due to its low interfacial and active-site
affinity for zwitterionic phospholipids, has low activity to release
fatty acids from mammalian cells and cannot effectively elicit
eicosanoid formation from unstimulated neutrophils. In contrast,
exogenous hsPLA2-V can effectively hydrolyze phospholipids
in the outer plasma membranes of mammalian cells and can also induce a
significant degree of eicosanoid formation from unstimulated neutrophils.
sPLA2s, including hsPLA2-V, have little
sn-2 arachidonoyl specificity because their active sites can
hold only about nine carbons in the sn-2 acyl chain (38).
Thus, high activity of hsPLA2-V to elicit eicosanoid
formation should derive from its ability to bind to the outer plasma
membrane and release various fatty acids from membrane phospholipids
including arachidonic acid. This notion is consistent with a recent
finding that exogenous arachidonic acid is rapidly transported across
the neutrophil plasma membrane via a protein-facilitated mechanism
(39). Due to the inability of hsPLA2-IIa to directly act on
intact cell membranes, several activation mechanisms have been proposed
to account for its interactions with cells. They include membrane perturbation, including cell surface exposure of aminophospholipids (e.g. phosphatidylserine and -ethanolamine) (40), prior
activation of cPLA2 (4, 5), and binding to cell surfaces
via heparinoids (41). Our results would suggest that none of these
activation steps are essential for the action of hsPLA2-V
on CHO-K1, RAW264.7, and human neutrophils, thereby suggesting that
under the physiological conditions, hsPLA2-V can
effectively act on unprimed mammalian cells. The precise physiological
significance of the action of hsPLA2-V on neutrophils was
not elucidated in this investigation. Neutrophils are not known to have
high sPLA2 activities. However, the close proximity of
these cells to mast cells and macrophages, which secrete
sPLA2s, including hsPLA2-V during many
inflammatory processes (10, 11), and the ability of
hsPLA2-V to elicit substantial LTB4 synthesis
and secretion in nanomolar concentrations, suggest a possible paracrine
mechanism of neutrophilic inflammation. Further studies are required to
elucidate the specific physiological role of this unique
PLA2 isoform in granulocytic inflammation.