From the
Endothelial nitric oxide synthase (eNOS) is a calmodulin
(CaM)-dependent, membrane-associated, myristoylated enzyme, which has
an important role in regulation of vascular tone and platelet
aggregation. In this study, wild-type and mutant forms of bovine eNOS
were overexpressed in a baculovirus/Sf9 insect cell system and examined
for interactions with membrane phospholipids. Purified wild-type eNOS
binds to pure anionic phospholipid vesicles but not to neutral
phospholipid vesicles, demonstrating that eNOS attachment to lipid
bilayers requires electrostatic as well as hydrophobic interactions.
Moreover, catalytic activity of the enzyme is potently inhibited by
anionic phospholipids, notably phosphatidylserine (PS), but not by
neutral phospholipids. eNOS activity is also significantly inhibited
upon enzyme binding to biological membranes isolated from cultured
cells. Binding of eNOS to PS vesicles prevents subsequent binding of
the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not
affected by site-specific mutation of the myristic acid acceptor site
in the enzyme. Deletional mutation of the eNOS CaM-binding domain,
however, results in loss of binding capacity of the enzyme not only for
CaM-Sepharose but also for PS vesicles. Furthermore, removal of the
CaM-binding domain converts eNOS from a membrane to a cytosolic protein
when the enzyme is expressed in Sf9 cells. These data demonstrate that
electrostatic interactions between anionic membrane phospholipids and
basic residues in the eNOS CaM-binding domain are important for enzyme
membrane association. Membrane association can thus function to inhibit
eNOS catalytic activity by interfering with the interaction of the
enzyme with calmodulin.
Nitric oxide is synthesized in diverse mammalian tissues by
three different isoforms of nitric oxide synthase
(NOS)
eNOS is found predominately in
membrane (rather than cytosolic) subcellular fractions of endothelial
cells(5, 6) . Furthermore, since the distribution of
eNOS activity in various membrane fractions closely resembles that of
the plasma membrane marker, 5`-nucleotidase, it has been suggested that
most, if not all, eNOS in endothelial cells is specifically associated
with the plasma membrane(6) . Thus, it is speculated that
membrane association of eNOS may have a role in coupling the
enzyme's activation to cell surface receptors or to physical
stimuli such as hemodynamic fluid shear stress. It has further been
suggested (7, 8, 9, 10) that membrane
association of eNOS is due to the presence of an N-myristoylation consensus sequence (MGXXXS), a
sequence not present in either iNOS or nNOS. Site-directed mutagenesis
of the myristic acid acceptor site (Gly-2) converts eNOS from a
membrane to a cytosolic protein in transfected COS-7
cells(7, 8) .
Myristoylation may not be the only
mechanism responsible for membrane attachment of eNOS. The Gibbs free
energy for binding of a myristoylated peptide to a phospholipid vesicle
is 8 kcal/mol, equivalent to an apparent K
eNOS expressed in Sf9
cells was similar to the BAEC enzyme in terms of its fatty acylation by
myristate and palmitate(9, 26) . Sf9 cells were
incubated with [
(
)(1) . In blood vessels, endothelial
NOS (eNOS) plays a key role in regulation of vascular tone and platelet
aggregation. Neuronal NOS (nNOS) is involved in neurotransmission in
the brain and peripheral nervous system, while the cytokine-inducible
NOS (iNOS) is an important mediator of the tumoricidal and bactericidal
actions of macrophages. Each of the three NOS isoforms is similar in
structure and function and catalyzes the oxidation of L-arginine to produce NO and L-citrulline via a
complex reaction involving NADPH, FAD, FMN, tetrahydrobiopterin, and a
p450-type heme moiety(2) . Primary structures of each of the
enzymes contain conserved consensus sequences for binding of heme,
calmodulin (CaM), FMN, FAD, and NADPH (3). The presence of a putative
CaM-binding domain in iNOS is intriguing, since iNOS (in contrast to
eNOS and nNOS) is a Ca
-independent enzyme. CaM
appears to function as a subunit of iNOS, however, remaining tightly
bound to the enzyme even in the absence of elevated intracellular
Ca
(4) .
of 100 µM(11) . Myristoylation alone,
therefore, is probably not sufficient for stably anchoring a protein to
a cellular membrane(12) . Indeed, many myristoylated proteins
are cytosolic(13) . Myristoylated proteins that are
membrane-associated often interact with membranes, not only through
hydrophobic interactions between protein and membrane fatty acyl
chains, but also through interactions of positively charged protein
residues with negatively charged membrane phospholipids. Furthermore,
it appears that a substantial fraction of both neuronal NOS (14, 15) and macrophage-inducible NOS (16) is
also localized to membranes. Since these enzymes lack an N-myristoylation site, it is clear that properties of NOS
other than myristoylation can mediate enzyme membrane association.
Therefore, in this study we have investigated in detail the
interactions of the eNOS enzyme with phospholipids present in membranes
and have determined the functional consequences of these interactions
on enzyme catalytic activity.
Materials
Spodoptera frugiperda Sf9
cells, baculovirus transfer vector pVL1393, and Baculogold viral DNA
were purchased from Pharmingen (San Diego, CA). Grace's insect
cell culture medium was obtained from Life Technologies, Inc.
2`,5`-ADP-Sepharose and CaM-Sepharose were obtained from Pharmacia
Biotech Inc. Phospholipids were purchased from Avanti Polar Lipids,
Inc. (Alabaster, AL). AG 50W-X8 cation exchange resin, electrophoresis
reagents, and Bradford protein dye reagent came from Bio-Rad. L-[C]Arginine,
[
H]myristic acid,
[
H]palmitic acid, and ECL detection kit were
purchased from Amersham Corp, and EN
HANCE was purchased
from DuPont NEN. Anti-eNOS antibody was a product of Transduction
Laboratories (Lexington, KY), and Sequenase 2.0 was a product of U. S.
Biochemical Corp. All other chemicals were obtained from Sigma.
Expression, Purification, and Characterization of
eNOS
The cDNA sequence encoding bovine eNOS (17) was
subcloned into the EcoRI site of the transfer vector pVL1393.
Recombinant transfer vector was then cotransfected with Baculogold
viral DNA into Sf9 insect cells, and a high titer recombinant viral
stock was obtained and used for subsequent infection of Sf9 cells.
Insect cells were infected at a multiplicity of infection of 5 in
Grace's medium supplemented with hemin chloride (3 µg/ml).
Three days after infection, cells were harvested and lysed on ice for
30 min in 50 mM Tris-HCl (pH 7.0) buffer containing protease
inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml
each antipain, aprotinin, bestatin, leupeptin, soybean trypsin
inhibitor, and pepstatin A), 0.1% 2-mercaptoethanol, and 1% Triton
X-100 (Buffer A). Lysate was applied to a 2`,5`-ADP-Sepharose affinity
column. The column was washed with 20 volumes of Buffer B (Buffer A
containing 20% glycerol but without Triton X-100), 20 volumes of Buffer
B containing 0.5 M NaCl and 2 mM EGTA, and an
additional 20 volumes of Buffer B alone. eNOS was eluted from the
column with 10 mM NADPH in Buffer B. Following chromatography
on 2`,5`-ADP-Sepharose, eNOS protein was >95% pure as determined by
Coomassie staining of purified proteins separated on SDS-polyacrylamide
gels. Fatty acyl modifications of eNOS in Sf9 cells were detected by
radiolabeling of cells with either [H]myristate
or [
H]palmitate. 5 h after addition of 1 mCi of
either compound, Sf9 cells were harvested and eNOS was purified by
2`,5`-ADP-Sepharose. Purified proteins were separated on
SDS-polyacrylamide gels, and gels were washed overnight in either 1 M Tris-HCl or 1 M hydroxylamine (pH 7.0). Radiolabel
remaining in the gel was visualized by fluorography with
EN
HANCE.
Cosedimentation of eNOS with Phospholipid Vesicles and
Biological Membranes
Phospholipids in chloroform solution were
dried to a thin film, resuspended at a concentration of 5 mg/ml in 20
mM Tris-HCl (pH 7.5), and sonicated with a probe-type
sonicator for 5 min. This method of phospholipid vesicle preparation
yields a mixture of large unilamellar and multilamellar
vesicles(18) . Vesicles were also prepared from biological
membranes. Bovine aortic endothelial cells (BAEC) and HeLa cells were
grown to confluence, harvested, and homogenized in 50 mM
Tris-HCl buffer (pH 7.5) containing protease inhibitors and 0.1%
2-mercaptoethanol. Unbroken cells were pelleted at 1,000 g for 10 min. Membranes in the supernatant were then pelleted by
centrifugation at 100,000
g for 30 min. The pellet was
resuspended in buffer containing 1 M KCl and recentrifuged at
100,000
g. The final membrane pellet was resuspended
in Buffer B and stored at -70 °C. Purified eNOS (1 µg)
and either pure phospholipid vesicles (200 µM) or membrane
vesicles (equivalent to 50 µg of protein) were mixed in a volume of
500 µl in Buffer B containing 100 mM NaCl. Mixtures were
centrifuged at 100,000
g for 30 min at 35 °C.
Pellets were resuspended in 500 µl Buffer B and analyzed with
supernatants on SDS-polyacrylamide gels followed by Coomassie staining.
For binding curves, PS vesicles (200 µM) were mixed with
varying amounts of wild-type and (-myr)eNOS (1-36 µg)
and binding was measured by Bio-Rad protein assay of pellets after
centrifugation.
Phospholipid Vesicle and Membrane Vesicle Effects on eNOS
Activity
Purified eNOS (1 µg) was mixed with varying amounts
of pure phospholipid vesicles (0-500 µM), and
activity was determined by the method of Bredt and Snyder(19) ,
which determines the rate of formation of L-[C]citrulline from L-[
C]arginine (100 µM) in
the presence of excess cofactors including Ca
(2
mM), CaM (200 units), NADPH (1 mM), FAD (4
µM), FMN (4 µM), and tetrahydrobiopterin (40
µM). Product (L-[
C]citrulline) was separated from
substrate on Bio-Rad AG 50W-X8 cation exchange columns. Biological
membrane effects on eNOS activity were determined by incubation of the
enzyme (1 µg) with membranes (50 µg of protein) followed by
centrifugation of the mixture at 100,000
g for 30 min
and assay of arginine-to-citrulline conversion activity in the
resuspended membrane pellet. In some enzyme activity determinations,
the products that were detected were the stable degradation products of
NO, quantitated by a chemiluminescence NO analyzer.
CaM-Sepharose Chromatography of eNOS
Binding
capacities of various forms of eNOS for CaM was determined by
CaM-Sepharose affinity chromatography. Purified eNOS (4 µg) was
applied to a CaM-Sepharose column (1 ml), and the column was washed
extensively with Buffer B containing 2 mM CaCl.
Bound eNOS was specifically eluted from the column with Buffer B
containing 2 mM EGTA.
PCR-based Mutagenesis of eNOS
Mutations were
introduced into the wild-type bovine eNOS cDNA sequence (17) by
the splicing-by-overlap-extension technique(20) . In the
(-myr) mutant, the glycine 2 codon (GGC) was mutated to an
alanine codon (GCC). In the CaM mutant, 60 nucleotides (coding for
eNOS residues 493-512) were deleted. Incorporation of the desired
mutations was confirmed by dideoxy chain termination sequencing with
Sequenase 2.0.
Subcellular Localization of Wild-type and Mutant Forms of
eNOS Expressed in Sf9 Insect Cells
Sf9 cells were infected with
recombinant baculovirus encoding either wild-type eNOS,
(-myr)eNOS, or CaM eNOS. Three days after infection, cells
were harvested and homogenized in 50 mM Tris-HCl buffer (pH
7.5) containing protease inhibitors and 0.1% 2-mercaptoethanol.
Unbroken cells were pelleted at 1,000
g for 10 min and
the first supernatant was then centrifuged at 100,000
g for 30 min to obtain particulate (pellet) and cytosolic
(supernatant) fractions. Following resuspension of the pellet to the
original volume of the homogenate, equal volumes of supernatant and
pellet fractions were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting with anti-eNOS antibody. Bound
antibody was visualized with the ECL detection system and
autoradiography. Relative amounts of eNOS in the different fractions
was quantitated by densitometry of autoradiograms.
Expression, Purification, and Characterization of
Wild-type eNOS
The purpose of this investigation was to
characterize the interactions between eNOS and membrane phospholipids
and to determine the consequences of these interactions on eNOS
activity. Comprehensive study of eNOS-phospholipid interactions,
however, requires larger quantities of purified protein than can be
routinely obtained from cultured endothelial cells(21) .
Therefore, we developed a baculovirus/Sf9 insect cell system for
overexpression of the bovine eNOS enzyme. Expression of heme-containing
enzymes in a baculovirus system often requires hemin supplementation of
the culture medium for expression of protein with high specific
activity(22) . This requirement has recently been demonstrated
for baculovirus overexpression of nNOS(23, 24) . In the
present study, addition of 3 µg/ml hemin chloride to the insect
cell culture medium resulted in production of eNOS enzyme having a
3.3-fold higher specific activity than enzyme produced in the absence
of hemin supplementation. Expressed protein was purified to >95%
homogeneity by affinity chromatography on
2`,5`-ADP-Sepharose(19) . As shown in , most
properties of the purified overexpressed enzyme, including subunit
molecular mass, k for L-arginine, Ca
and cofactor dependence, and
inhibition by substrate analog inhibitors were identical to those
previously reported for the BAEC enzyme(21) . The specific
enzyme activity (146 nmol of L-citrulline produced/mg/min)
determined by L-arginine to L-citrulline conversion
was 10-fold higher than that reported previously. Similar specific
activity was also determined using measurements of
NO
and NO
(quantitated by chemiluminescence after vanadium/HCl reduction).
The explanation for the higher enzyme activity in the present study
compared to that reported previously remains unclear. It is likely that
the ability to purify the baculovirus-expressed enzyme in highly
concentrated solution avoids the thermal denaturation and inactivation
characteristic of more dilute enzyme(25) . In addition,
purification by a single chromatography step allows for assay of
purified protein within a few hours after beginning the purification
procedure with less time for loss of activity.
H]myristic acid, eNOS was
purified, and incorporation of radiolabel into the protein was analyzed
by fluorography of proteins separated on SDS-polyacrylamide gels.
[
H]Myristate was incorporated into the protein
and was stable to hydroxylamine treatment (data not shown), suggesting
that the lipid is attached to the protein via an amide linkage. Sf9
cells were also labeled with [
H]palmitate, and
acylation was determined by the same protocol used to study
myristoylation. Radioactive palmitate was also incorporated into eNOS
but was quantitatively removed by hydroxylamine treatment (data not
shown), suggesting that lipid attachment occurs through an ester bond.
Binding of eNOS to Phospholipid Vesicles
The
interaction of eNOS with specific membrane phospholipids was assessed
by cosedimentation analysis. Phospholipid vesicles (each containing
identical dioleoyl fatty acyl side chains) were formed from each of the
eight most abundant membrane phospholipids(27) . These included
the anionic (acidic, negatively charged) lipids phosphatidylserine
(PS), phosphatidic acid (PA), cardiolipin (CL), and
phosphatidylinositol (PI) and the neutral (zwitterionic) lipids
phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin
(SM), and lysophosphatidylcholine (LPC). Vesicles (200 µM)
were mixed with purified eNOS (1 µg) and centrifuged at 100,000
g. Supernatant and pellet fractions were analyzed by
SDS-polyacrylamide gel electrophoresis and Coomassie staining. eNOS
selectively bound to phospholipid vesicles formed from anionic
phospholipids (Fig. 1A) but not to vesicles formed from
neutral phospholipids (Fig. 1B). Cosedimentation
represented specific binding rather than aggregation or precipitation,
since all minor contaminating proteins in the preparation remained in
the supernatant fraction. Binding was Ca
-independent,
since no change in sedimentation was observed in the presence of 2
mM EGTA. Selective binding of eNOS to negative lipids (but not
to neutral lipids) clearly demonstrates that eNOS attachment to
membrane lipids requires electrostatic interactions in addition to
hydrophobic interactions. Thus, although myristoylation (7-10)
and perhaps also palmitylation (26) may be required for eNOS
membrane phospholipid binding, these modifications by themselves are
not sufficient to completely account for the association. If fatty
acylation alone were sufficient for binding, the enzyme should bind
equally well to both neutral and negatively charged phospholipid
vesicles.
Figure 1:
Cosedimentation analysis of
eNOS binding to phospholipid vesicles. Purified wild-type eNOS (1
µg) was mixed with 200 µM each of phospholipid
vesicles formed from PS, PA, CL, PI, PC, PE, SM, or LPC. After
centrifugation at 100,000 g, supernatant (S)
and pellet (P) fractions were analyzed on Coomassie-stained
SDS-polyacrylamide gels. Only the relevant portion of the gel is shown. A, anionic phospholipids; B, neutral phospholipids.
Similar results were obtained in three separate
experiments.
Effect of Phospholipids on eNOS
Activity
Phospholipid vesicles were also tested for their
effects on eNOS catalytic activity. Anionic phospholipids (PS, PA, CL,
and PI) inhibited enzyme activity with IC values of
approximately of 6, 50, 45, and 350 µM, respectively (Fig. 2B). Neutral phospholipids (PC, PE, SM, and LPC)
were completely without effect (Fig. 2A). Inhibition
appears to be correlated with binding, since PS, PA, and CL vesicles
demonstrated complete inhibition and complete binding while PI showed
partial inhibition and partial binding. PS inhibition also appears to
depend on arrangement of the phospholipids into a bilayer, since
inhibition was completely reversed by subsequent addition of the
detergents Triton X-100 (0.1%) and CHAPS (2 mM). The fact that
full activity of PS-inhibited eNOS could be restored by subsequent
addition of detergent demonstrates that inhibition is not simply a
result of PS-promoted denaturation of the enzyme. If inhibition were
due to denaturation, it would not be reversible. In order to confirm
that the observed inhibition was not an artifact of baculovirus
expression, eNOS was purified from BAEC(21) . eNOS purified from
BAEC was also potently inhibited by PS. Furthermore, solubilization of
BAEC particulate fractions with Triton X-100 resulted in an
approximately 4-fold increase in eNOS activity (specific activity was
increased from 0.218 nmol/mg/min to 0.881 nmol/mg/min) confirming the
observation previously made by Pollock et al.(21) , who
found a >4-fold increase in BAEC particulate eNOS activity following
CHAPS solubilization. Detergent solubilization of particulate fractions
apparently releases the enzyme from inhibitory interactions with
anionic phospholipids. PS inhibition of eNOS probably has physiological
significance, since the enzyme functions in endothelial cells in close
association with the PS-rich inner leaflet of the plasma membrane. The
inner leaflet of the endothelial cell plasma membrane may in fact have
a PS content that approaches 50% of total inner leaflet phospholipid.
This conclusion follows from the following two observations. First,
whereas PS represents only 5% of total endothelial cell phospholipid
(28), it is found in mammalian cells almost exclusively in plasma
membranes, which comprise 20% of the total cellular membranes.
Intracellular membranes, representing the other 80% of cellular
membranes, are almost completely devoid of PS(29) . Endothelial
plasma membranes, therefore, may have a total PS content of up to 25%.
Furthermore, endothelial cells and other vascular cells such as
erythrocytes and platelets possess a lipid membrane asymmetry with all
of the PS being segregated in the inner leaflet(30) .
Aminophospholipid translocases (``flippases'') maintain an
antithrombogenic surface on vascular cells by selectively transporting
PS from the outer to the inner leaflet, which could thus consist of as
much as 50% PS. Membrane association of eNOS, therefore, may hold the
enzyme in an (at least partially) inactive state.
Figure 2:
Effect of phospholipids on eNOS activity.
Purified wild-type eNOS (1 µg) was mixed with 0, 10, 50, 100, 200,
and 500 µM phospholipid vesicles formed from PC, PE, SM,
LPC, PS, PA, CL, or PI. eNOS activity was then determined by monitoring
the rate of conversion of L-arginine to L-citrulline. A, neutral phospholipids; B, anionic phospholipids.
Results shown are representative of three
experiments.
PS Interactions of eNOS Lacking the N-Myristoylation
Site
Lack of binding of eNOS to neutral phospholipid vesicles
suggests that fatty acid modifications (such as N-myristoylation) are not sufficient for attachment of the
protein to lipid bilayers. Binding to anionic phospholipid vesicles,
however, may require both electrostatic interactions and hydrophobic
insertion of myristate into the membrane interior. In order to further
define the role of eNOS N-myristoylation in binding to (and
inhibition by) anionic phospholipids, a mutant of eNOS lacking the N-myristoylation site was expressed in the baculovirus system.
PCR-based mutagenesis was used to replace the glycine 2 codon (GGC)
with an alanine codon (GCC). The (-myr) mutant protein was
expressed in Sf9 cells and purified by 2`,5`-ADP-Sepharose
chromatography. Sf9 cells expressing either wild-type or mutant protein
were treated with [H]myristate. Radiolabel was
incorporated only into wild-type protein. No labeling was detected for
the (-myr) mutant (data not shown). Specific activities of
purified wild-type and mutant proteins were determined and found to be
identical, indicating that myristoylation has no influence on activity
of the isolated protein. Both proteins were also inhibited to the same
extent by PS vesicles. To determine the apparent affinity of wild-type
and (-myr)eNOS for PS vesicles, binding of varying amounts of the
protein (1-36 µg) to 200 µM PS vesicles was
quantitated and analyzed by the method of Scatchard(31) .
Saturable binding curves (Fig. 3, panelsA and C) and apparent dissociation constants (K
values) determined by Scatchard analysis (Fig. 3, panelsB and D) were very similar for the
two proteins (Fig. 3). K
values for
binding of wild-type and (-myr)eNOS were 1.0 and 1.5
µM, respectively, indicating that myristoylation is not
required for eNOS binding to pure PS vesicles. Binding of eNOS to
biological membrane vesicles prepared from BAEC, CHO cells, and COS-7
cells, however, has been shown previously to be dependent on enzyme
myristate modification(10) . The present study suggests that the
requirement for a hydrophobic component in the phospholipid interaction
becomes less important when the percent anionic lipid in the
phospholipid vesicle is increased to 100% of the total.
Figure 3:
Binding of wild-type eNOS and
(-myr)eNOS to PS vesicles. PanelsA and C, wild-type and (-myr)eNOS was incubated with PS
vesicles and binding was determined as described under
``Experimental Procedures.'' PanelsB and D, binding data plotted according to Scatchard where protein
bound is expressed as pmol of eNOS bound/nmol of phosphatidylserine.
Results are representative of two
experiments.
Effects of PS Binding on eNOS-CaM Interactions
PS
binding of eNOS appears to result in enzyme inhibition. Because eNOS is
a CaM-dependent enzyme, we have considered the possibility that eNOS
may be regulated similarly to several other proteins that are regulated
by both CaM and PS. These include the brain proteins,
neuromodulin(32) , neurogranin(33) ,
calcineurin(34) , and NAP-22(35) , as well as intestinal
brush border myosin I (36) and the ubiquitously expressed MARCKS
(myristoylated alanine-rich protein kinase C substrate)
protein(37) . All of these proteins are similar in that they
possess a CaM-binding domain containing clusters of basic residues,
which also bind anionic phospholipids, notably PS, in the plasma
membrane. CaM-binding domains in proteins are typically 20 residues in
length and conform to the Baa-helical (basic amphiphilic -helix)
model with hydrophobic residues on one side of the helix and positively
charged amino acids on the opposing face(38) . Bovine eNOS
contains such a sequence at positions 493-512 of the polypeptide
chain(17) . We have investigated the hypothesis that binding of
PS to this domain in eNOS interferes with subsequent CaM binding. eNOS
(4 µg) was preincubated with 500 µM of either PS
vesicles or PC vesicles (as a control) for 10 min at 37 °C and then
subjected to affinity chromatography on CaM-Sepharose. eNOS
preincubated with PC vesicles bound to CaM-Sepharose in the presence of
Ca
and was specifically eluted with EGTA (Fig. 4A). In contrast, eNOS preincubated with PS
vesicles did not bind to CaM-Sepharose and came through the column in
the flow-through fractions (Fig. 4B). Loss of eNOS
binding to CaM-Sepharose in these experiments appears to be due to
binding of PS to the eNOS CaM-binding domain, rather than due to an
effect of PS to significantly alter eNOS three-dimensional structure.
This interpretation is supported by experiments in which PS
preincubation was found to have no effect on subsequent binding of eNOS
to 2`,5`-ADP-Sepharose. Binding of eNOS to 2`,5`-ADP (an NADPH analog)
would probably not occur with a denatured eNOS enzyme. PS bound to eNOS
did not appear to be displaced by CaM under these conditions. A similar
inability of CaM to displace previously bound PS was also observed in
the cosedimentation experiments. Three-fold molar excess concentrations
of CaM (relative to eNOS) when added after preincubation with PS
(4200-fold molar excess relative to eNOS) did not inhibit
cosedimentation of eNOS with PS vesicles, nor was any CaM detected in
the pellet fraction following sedimentation (data not shown). However,
when eNOS was first incubated with a 3-fold molar excess of
Ca
and CaM (10 min at 37 °C), subsequent
cosedimentation with PS vesicles was reduced by approximately 70%. PS
and CaM thus appear to compete for binding to the same site in the
wild-type eNOS enzyme. Taken together, these data suggest that PS may
inhibit eNOS activity by binding to the CaM-binding domain of the
protein, thus preventing the calmodulin-protein interaction that is
required for activation of the enzyme.
Figure 4:
CaM-Sepharose chromatography of eNOS
following preincubation with PC or PS vesicles. Purified wild-type eNOS
(4 µg) was mixed with 500 µM PC or PS vesicles and
preincubated for 10 min at 37 °C. Mixtures were applied to
CaM-Sepharose columns and four fractions (1 ml each) were eluted with
buffer containing 2 mM Ca (flow-through).
Four additional fractions (1 ml each) were then eluted with buffer
containing 2 mM EGTA. Fractions were vacuum-evaporated down to
50 µl and proteins were analyzed on SDS-polyacrylamide gels with
Coomassie staining. A, eNOS preincubated with PC vesicles; B, eNOS preincubated with PS vesicles. Equivalent results were
obtained in three experiments.
PS Interactions of eNOS Lacking the Putative CaM-binding
Domain
Loss of eNOS interaction with CaM-Sepharose following
preincubation with PS suggests that PS and CaM bind to the same site on
the enzyme. To further investigate this hypothesis, we created a
deletion mutant of eNOS by PCR-based mutagenesis in which the putative
CaM-binding domain (TRKKTFKEVANAVKISASLM, residues 493-512) was
removed. The mutant protein (designated CaM eNOS) was expressed in
the baculovirus system and purified by 2`,5`-ADP-Sepharose affinity
chromatography.
CaM eNOS comigrated with wild-type eNOS on
SDS-polyacrylamide gels and reacted with anti-eNOS antibody on Western
blots. The
CaM mutant, however, had no capacity to catalyze the
conversion of L-arginine to L-citrulline. Moreover,
deletional mutation resulted in a complete loss of CaM-binding capacity
of the enzyme. Wild-type and
CaM eNOS were subjected to affinity
chromatography on CaM-Sepharose. Wild-type eNOS bound to CaM-Sepharose
in the presence of Ca
and was specifically eluted
with EGTA (Fig. 5A).
CaM eNOS, on the other hand,
did not bind to CaM-Sepharose and came through the column in the
flow-through fractions (Fig. 5B). Residues 493-512
of eNOS, previously identified as a CaM-binding domain solely on the
basis of sequence analysis, thus appear to represent a true CaM-binding
site. Furthermore, this domain also functions as a PS-binding site.
Cosedimentation analysis of wild-type and
CaM eNOS showed that
only the wild-type enzyme (and not the mutant) cosedimented with PS
vesicles (Fig. 6). Loss of binding of
CaM eNOS to PS
vesicles (similar to the loss of PS binding that occurred with
wild-type eNOS after preincubation with Ca
/CaM) was
probably not due to conformational changes in the protein, which could
result from a deletional mutation. If major changes in protein folding
had occurred, the enzyme would probably have lost its binding capacity
for 2`,5`-ADP-Sepharose as well.
Figure 5:
CaM-Sepharose chromatography of wild-type
eNOS and CaM eNOS. Purified wild-type eNOS or
CaM eNOS (4
µg each) were applied to CaM-Sepharose columns and four fractions
(1 ml each) were eluted with buffer containing 2 mM
Ca
(flow-through). Four additional fractions (1 ml
each) were then eluted with buffer containing 2 mM EGTA.
Fractions were vacuum-evaporated down to 50 µl and proteins were
analyzed on SDS-polyacrylamide gels with Coomassie staining. A, wild-type eNOS; B,
CaM eNOS. Results are
representative of three experiments.
Figure 6:
Cosedimentation analysis of binding of
wild-type eNOS and CaM eNOS to PS vesicles. Purified wild-type
eNOS or
CaM eNOS (1 µg each) were mixed with 200 µM PS vesicles. After centrifugation at 100,000
g,
supernatant (S) and pellet (P) fractions were
analyzed on Coomassie-stained SDS-polyacrylamide gels. Only the
relevant portions of the gel is shown. Similar results were obtained in
three experiments.
Interactions of eNOS with Biological
Membranes
Association of eNOS with anionic phospholipids appears
to be mediated, at least in part, through electrostatic interactions
between negative charges in phospholipid polar head groups and
positively charged amino acids in the eNOS CaM-binding domain. Among
the residues in bovine eNOS that may contribute to this interaction are
Arg-494, Lys-495, Lys-496, Lys-499, and Lys-506. To determine whether
the eNOS CaM-binding domain also has a role in enzyme membrane
association in intact cells, wild-type eNOS, (-myr)eNOS, and
CaM eNOS were expressed in Sf9 cells and the subcellular
localization of expressed proteins was determined. Cells were infected
under identical conditions used to produce enzyme for purification.
Three days after infection, cells were harvested and cytosolic and
membrane subcellular fractions were prepared. Fractions were analyzed
by SDS-polyacrylamide gel electrophoresis and Western blotting in order
to quantitate the relative amounts of expressed protein in the two
subcellular fractions. As shown in Fig. 7, wild-type eNOS was
found almost entirely in membrane fractions (91 ± 3%, mean
± S.D., n = 3) rather than cytosolic subcellular
fractions (9 ± 3%) of Sf9 cells. In contrast, (-myr)eNOS
was found almost entirely in cytosolic fractions (88 ± 4%)
rather than particulate fractions (12 ± 4%). Identical results
were also obtained when subcellular localization was quantitated by
assay of enzyme activity in soluble and detergent-solubilized
particulate fractions (data not shown). Similar subcellular
distribution of these two forms of eNOS has been demonstrated
previously in transfected COS-7 cells(7, 8) . The
present findings strongly suggest that the eNOS enzyme expressed in Sf9
cells is fully myristoylated. If only a subfraction of the enzyme were
myristoylated, wild-type eNOS would not be restricted to the
particulate fraction. Furthermore, myristoylation by itself is probably
not sufficient for eNOS membrane association. The CaM-binding domain of
the enzyme appears to also be required for stable membrane anchoring.
Thus,
CaM eNOS (which presumably is fully myristoylated) was found
predominately in cytosolic (65 ± 9%) rather than particulate (35
± 9%) subcellular fractions of Sf9 cells expressing the mutant
enzyme. Loss of membrane association of
CaM eNOS in Sf9 cells is
very likely due to loss of binding affinity of the deletional mutant
for anionic membrane phospholipids. An alternative explanation of these
results is that insect cells may lack a docking protein, which exists
in mammalian endothelial cells, that interacts with the eNOS
CaM-binding domain. This is very unlikely, however, since it has been
recently demonstrated by Busconi and Michel (10) that eNOS
binding to mammalian cell membranes does not involve a docking or
receptor protein. Treatment of biological membranes prepared from BAEC,
CHO cells, and COS-7 cells with either heat denaturation or
trypsinization thus has no effect on subsequent ability of treated
membranes to bind the wild-type eNOS enzyme(10) .
Figure 7:
Subcellular distribution of wild-type
eNOS, (-myr)eNOS, and CaM eNOS expressed in Sf9 cells. Sf9
cells were infected with recombinant baculovirus encoding either
wild-type eNOS, (-myr)eNOS, or
CaM eNOS. Cytosol and
membrane fractions from infected cells were prepared as described under
``Experimental Procedures.'' Relative amounts of eNOS protein
in fractions was quantitated by densitometry of Western blots. Results
shown are mean ± S.D. from three separate
infections.
The
functional effects of eNOS binding to biological membranes was also
determined. Wild-type enzyme was incubated with membrane vesicles
(heat-denatured to eliminate endogenous eNOS activity) prepared from
BAEC and HeLa cells. Membranes were sedimented at 100,000 g, and activity of the cosedimented (bound) enzyme was
determined. eNOS binding to BAEC and HeLa cell membranes reduced
catalytic activity by 71 ± 9% and 63 ± 11% (mean ±
S.D., n = 3), respectively. Membrane association thus
serves to significantly (although not completely) inhibit eNOS
activity. Partial (rather than complete) inhibition is not surprising,
since it is known that BAEC particulate fractions do have significant
enzyme activity, even in the absence of detergent solubilization.
Neutral lipids in membranes may thus diminish the inhibitory effects of
anionic lipids, as has been observed previously for other
phospholipid-regulated enzymes. The inhibitory effect of PI on actin
severing activity of adseverin, for example, is diminished by PC and
PE(39) . Similarly, PS activation of protein kinase C (perhaps
the best known of the phospholipid-regulated enzymes) is markedly
diminished by PC and SM (40). To examine the role of neutral
phospholipids in modulating the effects of anionic phospholipids on
eNOS activity, mixed phospholipid vesicles were formed from PC and PS
in which the PS content was varied from 0% to 100%. As shown in Fig. 8, inhibition of eNOS activity by these vesicles depended on
the percent anionic lipid in the vesicle, with half-maximal inhibition
occurring at approximately 40% anionic phospholipid.
Figure 8:
Effect of PC:PS mixed phospholipid
vesicles on eNOS activity. Purified wild-type eNOS (1 µg) was mixed
with phospholipid vesicles (200 µM) formed from mixtures
of PC and PS. eNOS activity was determined by assay of the rate of
conversion of L-arginine to L-citrulline. Results are
representative of three experiments.
Potential Role of Phospholipids in Regulation of eNOS in
Endothelial Cells
eNOS-phospholipid interactions may help to
explain a number of previous observations. For example, bradykinin
activation of eNOS in BAEC is associated with translocation of the
enzyme from membrane to cytosol(41) . It is conceivable that
translocation contributes to eNOS activation by releasing the enzyme
from inhibition by anionic membrane phospholipids. Activation of eNOS
also occurs in endothelial cells in response to acute increases in
fluid shear stress(42) . The mechanism of physiochemical signal
transduction in this circumstance is not known, but could involve
shear-induced eNOS subcellular translocation or shear-induced changes
in membrane fluidity (43) with resultant effects on
eNOS-phospholipid interactions. High affinity of eNOS for PS could also
function to specifically target the enzyme to the plasma membrane
(rather than to intracellular membranes), since PS is found almost
exclusively in cells in the plasma membrane(29) .
Table: Properties of purified bovine eNOS overexpressed
in a baculovirus system
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.