(Received for publication, November 14, 1994; and in revised form, June 9, 1995)
From the
The regulation of the lysophospholipase activity of the 85-kDa
cytosolic phospholipase A (PLA
) was studied in vitro and in stimulated macrophages. Bovine serum albumin
was found to inhibit lysophospholipase activity of the recombinant
85-kDa PLA
when assayed at a relatively low substrate
concentration. Inhibition could be reversed if the substrate
concentration was increased or if Ca
was present in
the assay. Incubation of recombinant enzyme with macrophage membranes
and lipid extracts from macrophage membranes resulted in the release of
arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that
with a bilayer substrate the PLA
can sequentially deacylate
the sn-2 then sn-1 acyl groups. This was verified by
demonstrating that the phospholipids, phosphatidylcholine and
phosphatidylinositol, were hydrolyzed to glycerophosphocholine and
glycerophosphoinositol by incubation with recombinant 85-kDa
PLA
. The 85-kDa enzyme was identified as the main
lysophospholipase activity in mouse peritoneal macrophage cytosols.
Addition of Ca
to the assay enhanced activity, but
this effect decreased as the substrate concentration was increased.
Incubation of macrophages with zymosan increased the lysophospholipase
activity of the 85-kDa PLA
in cytosols. Phosphorylation of
recombinant PLA
with mitogen-activated protein kinase
resulted in an increase in lysophospholipase, as well as, PLA
activity. In macrophages stimulated with zymosan release of
stearic acid (18:0) and palmitic acid (16:0) was observed in addition
to arachidonic acid (20:4). These results are consistent with a role of
the 85-kDa PLA
in regulating lysophospholipid levels in
macrophages during zymosan stimulation.
Lysophospholipids and arachidonic acid are produced by the
action of phospholipase A (PLA
) (
)on
membrane phospholipids during an inflammatory response. Arachidonic
acid can be metabolized to a number of potent proinflammatory
eicosanoids. In addition, arachidonic acid itself can act as a second
messenger and modulate a number of cellular functions(1) .
Lysophospholipids occur naturally at low levels (0.5-6%) in
membranes of unstimulated cells and are produced transiently during
phospholipid remodeling reactions (2, 3) . In
stimulated cells some of the 1-alkyl-linked lysophosphatidylcholine
produced is further metabolized into the inflammatory mediator platelet
activating factor(4) . Recent studies suggest that
lysophospholipids themselves can cause a variety of cellular responses
such as changes in intracellular Ca
and modulation of
chemotaxis(5, 6, 7) . In addition, at higher
concentrations lysophospholipids act as natural detergents and can
disrupt membrane structure(3) . Accumulation of
lysophospholipid can be controlled by reacylation to phospholipid or by
metabolism to water soluble glycerophosphocholine by
lysophospholipases. Multiple forms of lysophospholipases have been
identified and frequently coexist in cells, suggesting there may be
specific functional roles for individual
lysophospholipases(8) . However, little is known about the
mechanisms involved in lysophospholipase regulation.
The 85-kDa
PLA is a highly regulated enzyme that is now thought to
play an important role in mediating arachidonic acid release in
stimulated cells(9) . Interestingly, this enzyme also exhibits
a relatively high lysophospholipase activity and a low level of
transacylase activity(10, 11) . The lysophospholipase
and PLA
activities share one catalytic domain(12) .
Whether the 85-kDa PLA
acts as a lysophospholipase in
vivo and whether its lysophospholipase activity is regulated
during cell activation is not currently known. Simultaneous activation
of both PLA
and lysophospholipase activities of the 85-kDa
enzyme would provide an efficient mechanism for regulation of levels of
arachidonic acid and lysophospholipid. There is now substantial
evidence that both phosphorylation of the 85-kDa PLA
and
Ca
-dependent translocation to the membrane are
necessary for activation of the PLA
resulting in
arachidonic acid release(13) . In this study we have examined
whether the lysophospholipase activity of the 85-kDa PLA
is
regulated in zymosan-stimulated macrophages and whether it functions as
a lysophospholipase under physiological conditions.
Figure 1:
BSA inhibition of lysophospholipase
activity. Lysophospholipase activity was assayed in cytosols from Sf9
cells expressing recombinant PLA. Activity was assayed with
increasing concentrations of BSA (0-1.0 mg/ml) in the absence (1
mM EGTA) (open bars) or presence (striped
bars) of 5 mM Ca
. The data represent
the mean ± S.E. of three
experiments.
It
is well established that albumin has the capacity to bind
lysophospholipid and acts as a carrier of this lipid in the
blood(26) . To test the possibility that BSA was binding to the
lysophospholipid substrate and interfering with the lysophospholipase
assay, the enzyme was assayed in the presence of increasing amounts of
lysophospholipid at a fixed concentration of BSA (0.5 mg/ml) in the
absence of Ca (Fig. 2). As the substrate
concentration was increased from 50 to 200 µM, the degree
of inhibition by BSA decreased from 68 to 24%, indicating that
inhibition by BSA was dependent on the substrate concentration and
suggested that BSA was interacting with the substrate.
Figure 2: Effect of increasing lysophospholipid concentration on inhibition of lysophospholipase activity by BSA. Lysophospholipase activity was assayed in the absence (open bars) or presence (striped bars) of 0.5 mg/ml BSA using cytosol (0.4 µg) from Sf9 cells infected with recombinant virus. Control activity (100%) at each substrate concentration equals the activity in the absence of BSA. The percent hydrolysis was less than 6% at each substrate concentration. The data represent the mean ± S.E. of three experiments.
Figure 3:
Lysophospholipase activity in cytosols
from unstimulated and zymosan-stimulated macrophages. Macrophages were
incubated with (,
) or without zymosan (
,
)
for 30 min (30 particles/cell) and the cytosolic fraction prepared as
described under ``Experimental Procedures.''
Lysophospholipase activity was assayed at the times indicated in the
presence (
,
) or absence (
,
) of
Ca
. Activity was assayed using 10 µg of cytosolic
protein and 200 µM 1-palmitoyl-2-lyso-PC. Data from a
representative experiment run in triplicate is shown and expressed as
mean ± S.D.
Since mammalian cells contain multiple
lysophospholipases, it was necessary to determine that the
zymosan-stimulated lysophospholipase activity in macrophage cytosols
was due to the 85-kDa enzyme. When a neutralizing polyclonal antibody
against the recombinant baculovirus-expressed PLA was
incubated with purified recombinant PLA
, both PLA
and lysophospholipase activities of the enzyme were inhibited,
although a higher concentration of antiserum was required to inhibit
the lysophospholipase activity of the enzyme (Fig. 4). PLA
activity was completely inhibited at a serum dilution of 1:50,
whereas a 1:25 dilution of antiserum was required to inhibit
lysophospholipase activity by 82%. The two enzymatic activities share
one catalytic domain(12) , but the purified substrates for the
two different enzymatic activities assays differ structurally and
presumably in how they bind to the enzyme. Thus the differential
potency of the antiserum against the two enzymatic activities could
possibly be attributed to differences in the inhibition of the binding
of the substrates to the enzyme. When tested on macrophage cytosols,
preimmune serum did not inhibit activity, whereas immune serum
inhibited PLA
and lysophospholipase activity in both
unstimulated and zymosan-stimulated cytosols, verifying that these
activities were due to the 85-kDa PLA
(Fig. 5).
Lysophospholipase activity was similarly inhibited whether assayed in
the presence or absence of Ca
(data not shown).
Figure 4:
Inhibition of PLA and
lysophospholipase activities of the 85-kDa PLA
by
polyclonal antiserum. Recombinant PLA
was incubated with
different dilutions of serum from rabbits, before immunization (open bars) or serum from rabbits after immunization with
recombinant PLA
(striped bars), in 50 mM Tris, pH 7.6, containing 10% glycerol. Samples were then diluted
10-fold in the same buffer, and 25 µl (40 ng of enzyme) was assayed
for lysophospholipase (A) or PLA
activity (B) in a total volume of 50 µl. Data from a representative
experiment run in triplicate are shown and expressed as mean ±
S.D.
Figure 5:
Inhibition of PLA and
lysophospholipase activities in macrophage cytosols by polyclonal
antiserum to the 85-kDa PLA
. Macrophage cytosol from
unstimulated (open bars) and zymosan-stimulated (striped
bars) cells was incubated with a 1:50 dilution of preimmune (PI) or immune (I) serum from rabbits immunized with
recombinant PLA
. Samples were then assayed for
lysophospholipase (10 µg of cytosolic protein) (A) or
PLA
activity (20 µg of cytosolic protein) (B).
Data are expressed as a percent of the maximum response, which is the
activity in zymosan-stimulated cytosol incubated with preimmune serum
(100%). The data are from a representative experiment run in triplicate
and expressed as mean ± S.D.
Figure 6:
Separation of phosphorylated and
dephosphorylated forms of recombinant PLA by Mono Q
chromatography. Cytosols from Sf9 cells expressing recombinant
PLA
were prepared and treated with mock buffer (
,
) or phosphatase (
,
) as detailed under
``Experimental Procedures'' and loaded onto a Pharmacia Mono
Q HR 5/5 column equilibrated with buffer containing 230 mM NaCl. Fractions were eluted with a gradient from 230 to 400 mM NaCl at a flow rate of 0.5 ml/min and assayed for PLA
(
,
) or lysophospholipase activity (
,
) as described under ``Experimental
Procedures.''
Figure 7:
Rephosphorylation of PLA with
MAP kinase. Dephosphorylated recombinant PLA
purified by
Mono Q chromatography was incubated with purified MAP kinase as
described under ``Experimental Procedures.'' A,
samples were assayed for PLA
(open bars) and
lysophospholipase activity (shaded bars), and the data are
expressed as percent of control activity in the absence of MAP kinase
and represents mean ± S.E. of three experiments. B,
samples were also immunoblotted using a polyclonal antibody against the
recombinant PLA
.
Figure 8:
Release of fatty acids from macrophage
membranes incubated with recombinant PLA. Macrophage
membranes (20 µg of membrane protein) were incubated in the absence (open symbols) or presence (closed symbols) of
purified recombinant PLA
(50 ng) in 50 mM Tris, pH
7.6, with 5 mM CaCl
, 150 mM NaCl, and 100
µg/ml of fatty acid free BSA for the indicated times. The free
fatty acids released were analyzed by GCMS as described under
``Experimental Procedures.'' Data from a representative
experiment run in triplicate are expressed as mean ±
S.D.
In order to rule out a contaminating endogenous
lysophospholipase activity in the membrane, which hydrolyzed the
lysophospholipid supplied by the 85-kDa PLA activity, we
incubated membranes under the same conditions without the
PLA
, but with
1-[
C]palmitoyl-2-lyso-PC. Under these conditions
there was detectable lysophospholipase activity in the membranes (data
not shown), which might explain the slight increase in the release of
16:0 in membranes in the absence of PLA
. By Western
blotting we were able to detect low level contamination of the 85-kDa
PLA
in macrophage membranes washed with EDTA and EGTA (data
not shown), suggesting that the contaminating lysophospholipase
activity could be either the 85-kDa PLA
or another
lysophospholipase. When we heated these membranes at 65 °C for 90
min, we were able to completely abolish the endogenous
lysophospholipase activity in the membranes. Similar levels of fatty
acids were released from heat-treated membranes after incubation with
the 85-kDa PLA
as was released with untreated membranes
verifying that the release of 18:0 was due to the added 85-kDa enzyme
(data not shown). Additionally, we measured fatty acid release from
lipid extracts of macrophage membrane after incubation with PLA
(Fig. 9). As observed with the membrane substrate the
release of fatty acid was selective for 20:4 versus the other
fatty acids found at the sn-2 position. The release of 18:0
and 16:0 paralleled 20:4 release, plateauing at 1 h.
Figure 9:
Release
of fatty acids from lipid extracts of macrophage membranes incubated
with PLA. Lipid (0.7 µg of lipid phosphorus) extracted
from macrophage membranes was incubated in the absence (open
symbols) or presence (closed symbols) of purified
recombinant PLA
(50 ng) in 50 mM Tris, pH 7.6,
with 5 mM CaCl
, 150 mM NaCl, and 100
µg/ml of fatty acid free BSA for the indicated times. The free
fatty acids released were analyzed by GCMS as described under
``Experimental Procedures.'' Data from a representative
experiment run in triplicate are expressed as mean ±
S.D.
Figure 10:
Products released from
[H]choline-labeled PC and myo-[
H]inositol-labeled PI incubated
with PLA
. Recombinant PLA
(100 ng) was
incubated with liposomes of [
H]choline-labeled PC (A, B) or myo-[
H]inositol-labeled PI (C,
D) in the absence (A, C) or the presence (B, D)
of macrophage membrane lipid for the indicated times. Lipid products
(
, GPC;
, GPI;
, lyso-PC;
, lyso-PI) were
extracted and separated by TLC as described under ``Experimental
Procedures.'' Data from a representative experiment are expressed
as the mean of triplicate values.
Figure 11: Release of fatty acids from zymosan-stimulated macrophages. Macrophages were incubated without (open symbols) or with zymosan (30 particles/cell, closed symbols) for the indicated times in 1 ml of Dulbecco's modified Eagle's medium. The reaction was stopped by the addition of 0.5 ml of methanol. The samples were then analyzed for free fatty acids by GCMS as described under ``Experimental Procedures.'' The data are expressed as mean ± S.E. of three experiments.
Lysophospholipid levels must be tightly controlled due to
their deleterious effects on cellular membranes, and they increase only
transiently after cell activation. When macrophages are incubated with
lysophosphatidylcholine the majority is converted via the
lysophospholipase pathway to glycerophosphocholine rather than being
reacylated to phosphatidylcholine(7) . In the macrophage three
Ca-independent lysophospholipases have been
identified, the 85-kDa cytosolic PLA
and two others with
molecular masses of 27 and 28
kDa(10, 31, 32) . The functional role and
regulation of specific lysophospholipases in the cell is as yet
unknown. In neutrophils there appears to be an unidentified
lysophospholipase activity that can be stimulated during cell
activation(33) . In addition, during differentiation of HL-60
cells to neutrophil-like cells, there is an up-regulation of a 20-kDa
lysophospholipase(34) . In this study we show that zymosan
stimulation increases lysophospholipase activity in macrophage cytosol.
The 85-kDa PLA
is the predominant lysophospholipase and
PLA
activity in the cytosol, since both activities can be
completely abolished with specific antiserum. During zymosan
stimulation of macrophages, MAP kinase is activated and the 85-kDa
PLA
is phosphorylated, resulting in increased cytosolic
PLA
activity(14, 17) . Our data show that
the lysophospholipase activity can also be increased by phosphorylation
with MAP kinase.
While exploring assay conditions for assaying the
lysophospholipase activity of the 85-kDa enzyme, we observed that BSA
had an inhibitory effect on activity which could be reversed as the
substrate concentration was increased. In contrast, BSA increases
PLA activity by preventing enzyme trapping which occurs
when product accumulates in liposomes(24) . Albumin can bind
lysophospholipid as a monomer or can interact with lysophospholipid
micelles(35, 36) . In addition, it can remove
lysophospholipid bound to membranes and interacts with lysophospholipid
in liposomes to cause a disruption of membrane
integrity(37, 38) . One possibility for the inhibitory
effects of BSA is that it binds lysophospholipid, and at low substrate
concentrations there are predominately BSA-lysophospholipid complexes.
Only as the substrate concentration is increased is there
lysophospholipid substrate available for the enzyme.
We also
observed that Ca could abolish the inhibition of
lysophospholipase activity by BSA. The PLA
activity of the
85-kDa PLA
enzyme is Ca
-dependent.
Ca
does not play a role in catalysis, but is required
for binding of the enzyme to the phospholipid substrate, which is
mediated by a Ca
dependent phospholipid binding region (39, 40) . In contrast, the lysophospholipase activity
has been reported to be
Ca
-independent(10, 11, 16) .
This lack of Ca
dependence appears to only be unique
to the hydrolysis of lysophospholipid micelles. Hydrolysis of
lysophospholipid, when it was incorporated into a liposome substrate,
was Ca
-dependent, suggesting that the enzyme requires
Ca
to bind to a liposome, but not to a
lysophospholipid micelle(41) . Consequently, another
possibility is that BSA changes the structure of the lysophospholipid
substrate so that the enzyme now requires Ca
for
binding. It has also been observed that when assays are run in the
presence of Triton X-100 or glycerol that Ca
has a
stimulatory effect on activity (11) . More recently, Nalefski et al.(42) reported that the lysophospholipase
activity of the 85-kDa enzyme against lysophospholipid micelles was
Ca
dependent and the Ca
dependence
was more pronounced at lower substrate concentrations; however, in this
study the assays included BSA. Based on our observations, the
stimulatory effect of Ca
may have been due to the
reversal of the inhibition by BSA. It is of interest that we observed
that the lysophospholipase activity in macrophage cytosols was
stimulated by Ca
when assayed at low substrate
concentration, but was less affected by Ca
at higher
substrate concentrations. Possibly, the presence of cytosolic protein
interferes with the substrate, as was observed with the BSA, since
cytosolic proteins can also bind lysophosphatidylcholine(43) .
This effect was not observed in Sf9 cytosols, but the ratio of
PLA
to extraneous protein is much greater and the total
protein in the assay was 25-fold less than in assays run with
macrophage cytosols.
Hydrolysis of sn-2 fatty acids from
diacyl-linked cellular phospholipids by the 85-kDa PLA would generate substrate for the lysophospholipase activity of
this enzyme. Although 20:4 is enriched in ether-linked PC and PE pools,
in macrophages significant amount of acyl-20:4 pools exist that would
be a substrate for the arachidonate-preferring
PLA
(29, 30) . Lysophospholipids generated
in membranes can be reacylated or hydrolyzed by lysophospholipases,
resulting in the release of sn-1 fatty acid. Of the total 18:0
present in the PE and PC pools (both the sn-1 and sn-2 positions) of macrophages, 95 and 86% is found in the sn-1 position, respectively(30) . When fatty acid
release was measured from macrophages after stimulation with zymosan we
saw an increase in the release of 18:0 consistent with a role of
lysophospholipase activation during zymosan stimulation, although we
cannot rule out direct release of fatty acid by a phospholipase A
or other complex pathways. Also, we found that the 85-kDa enzyme
could catalyze release of 18:0 from macrophage membranes, which
occurred when apparent lysophospholipid levels accumulated to only a
small percentage of the total phospholipid present in the membrane. The
amount of lysophospholipid produced after incubation of membranes with
recombinant PLA
can be estimated based on the moles of 20:4
released assuming no reacylation of lysophospholipid occurs. At 1 h
when the release of 18:0 started to increase, the amount of
lysophospholipid was 0.7%, or 7 µM, of the amount of
phospholipid present (960 µM) in the membrane.
These
data suggest that the cPLA releases 18:0 from the sn-1 position through its lysophospholipase activity, although
we cannot rule out some release of 18:0 from the sn-2
position. However, this is unlikely since sn-2 saturated fatty
acids and even monoenoic fatty acids are poor substrates for the 85-kDa
PLA
(44, 45) . In addition the
lysophospholipase activity of the 85-kDa PLA
can hydrolyze
1-stearoyl-lyso-PC, although 1-palmitoyl-lyso-PC is a better
substrate(46) . Experiments measuring the production of
lysophospholipid and glycerophosphocholine or glycerophosphoinositol,
after incubation of the 85-kDa enzyme with PC and PI, confirmed that
the 85-kDa enzyme can completely deacylate diacyl phospholipid in a
bilayer. Particularly, the results with the diacyl substrate
stearoyl-arachidonoyl-PI show that levels of lysophospholipid are kept
low in the bilayer due to efficient conversion of the lysophospholipid
to GPI. In summary, the results suggest that the 85-kDa enzyme can
hydrolyze lysophospholipid as it accumulates in membranes, suggesting a
role for the lysophospholipase activity of the 85-kDa PLA
in regulating cellular lysophospholipid levels.