Identification of the Calmodulin-binding Domain of
Recombinant Calcium-independent Phospholipase A2
IMPLICATIONS FOR STRUCTURE AND FUNCTION*
Christopher M.
Jenkins,
Matthew J.
Wolf,
David J.
Mancuso, and
Richard W.
Gross
From the Division of Bioorganic Chemistry and Molecular
Pharmacology, Departments of Medicine, Chemistry, Molecular Biology
and Pharmacology, Washington University School of Medicine,
St. Louis, Missouri 63110
Received for publication, November 17, 2000
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ABSTRACT |
Calcium-independent phospholipase
A2 (iPLA2) is the major phospholipase
A2 activity in many cell types, and at least one isoform of
this enzyme class is physically and functionally coupled to calmodulin
(CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA2
(riPLA2
) underlying this interaction, multiple
techniques were employed. First, we identified calcium-activated CaM
induced alterations in the kinetics of proteolytic fragment generation
during limited trypsinolysis (i.e. CaM footprinting).
Tryptic digests of riPLA2
(83 kDa) in the presence of
EGTA alone, Ca+2 alone, or EGTA and CaM together
resulted in the production of a major 68-kDa protein whose kinetic rate
of formation was specifically attenuated in incubations containing CaM
and Ca+2 together. Western blotting utilizing antibodies
directed against either the N- or C-terminal regions of
riPLA2
indicated the specific protection of
riPLA2
by calcium-activated CaM at a cleavage site
15
kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of
riPLA2
. Second, functional characterization of products from these partial tryptic digests demonstrated that
90% of the 68-kDa riPLA2
tryptic product (i.e. lacking
the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the
presence of Ca2+, although >95% of the noncleaved
riPLA2
as well as a 40-kDa C-terminal peptide bound
tightly under these conditions. Third, when purified
riPLA2
was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide (694AWSEMVGIQYFR705) within the
15-kDa C-terminal fragment was identified by RP-HPLC, which bound to
CaM-agarose in the presence but not the absence of calcium ion. Fourth,
fluorescence energy transfer experiments demonstrated that this peptide
(694) bound to dansyl-calmodulin in a
calcium-dependent fashion. Collectively, these results
identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the
calcium-dependent regulation of iPLA2
by
CaM.
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INTRODUCTION |
The phospholipase A2-catalyzed release of arachidonic
acid from its phospholipid storage depots is a critical component of intra- and intercellular signal transduction. In most noncirculating cells (e.g. cardiac myocytes, pancreatic islet
-cells,
hippocampal neurons, and vascular smooth muscle cells),
calcium-independent phospholipase A2
(iPLA2)1 is the
major but not the only phospholipase A2 activity present (1-6). Multiple lines of experimental evidence implicate
iPLA2 as an important mediator of arachidonic acid release
in several cell types including: 1) the inhibition of the large
majority of AVP-induced arachidonic acid release in A-10 smooth muscle cells by 1-2 µM BEL (7); 2) the attenuation of the
release of arachidonic acid in lipopolysaccharide-stimulated
macrophages by either BEL or antisense DNA targeted to
iPLA2
(8, 9); 3) the robust release of ligand-stimulated
arachidonic acid in cells whose rise in cytosolic calcium ion content
is ablated by EGTA and
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl)ester (i.e. the release of
arachidonic acid occurs in the absence of Fura 2-detectable changes in
intracellular Ca+2 levels; Refs. 10 and 11); 4) the release
of arachidonic acid by agents that deplete intracellular
Ca+2 stores in the absence of receptor occupancy
(e.g. thapsigargin, cyclopiazonic acid, 2, 5-di-(t-butyl)-1,4-hydroquinone, and A23187) when
alterations in [Ca2+]i are prevented (10, 11);
and 5) the ionophore-induced release of arachidonic acid from HEK cells
expressing riPLA2
(but not in wild type HEK cells) with
its subsequent directed conversion to prostaglandin
E2 by cyclooxygenase I but not cyclooxygenase II (12).
To gain insight into the biochemical mechanisms responsible for the
activation of iPLA2 in stimulated cells, an early
observation was re-explored that demonstrated that the addition of
calcium ion to myocardial cytosol inhibited iPLA2 activity
(1). Subsequently, this inhibition was shown to be due to a protein
factor that was identified as calmodulin after purification to
homogeneity (13). These results were confirmed by reconstitution of
calcium-dependent iPLA2 inhibition utilizing
authentic calmodulin (13). Through detailed analysis of the interaction
of myocardial iPLA2 with CaM, we demonstrated the formation
of a catalytically inactive ternary complex of
CaM·Ca2+·iPLA2 that could be reversibly
dissociated by chelation of calcium ion with EGTA to regain full
catalytic activity. Subsequent work demonstrated that agents that
deplete intracellular calcium ion pools (e.g. thapsigargin
and cyclopiazonic acid), even in the absence of receptor occupancy or
alterations in cytosolic calcium ion concentration, resulted in
arachidonic acid release in intact cells (10). Furthermore, agents that
inhibited the interaction of calmodulin with its target proteins
through prevention of calcium-induced conformational changes in
calmodulin (e.g. W-7) resulted in the release of
[3H]arachidonic acid in resting A-10 smooth muscle cells
(10). These observations indicate that the majority of
iPLA2 activity in cells is tonically inhibited and that
dissociation of calmodulin from iPLA2 is the major
mechanism that transforms a calcium-independent catalytic activity into
an enzyme that is responsive to alterations in intracellular calcium
ion homeostasis. Collectively, these results gave rise to the
hypothesis of "calcium pool depletion-mediated iPLA2
activation" (10). To further define the physiologic relevance of this
hypothesis, it became important, from both a mechanistic perspective as
well as a therapeutic strategy, to determine the site(s) of interaction
of iPLA2
with calmodulin.
Since the first x-ray crystal structure (14) and NMR solution structure
(15) of CaM·peptide complexes became available, it was apparent that
calcium-induced conformationally activated calmodulin interacted with
its regulatory targets at multiple contact points. No universal
consensus sequences mediating the interaction of calmodulin with its
extremely diverse protein targets have been identified, although the
importance of positionally conserved hydrophobic and basic amino acid
residues has been demonstrated in some cases (16-20). Accordingly,
unambiguous identification of the loci of interaction of CaM with
iPLA2
cannot be recognized a priori from
comparisons of the iPLA2
primary sequence with other
known calmodulin-regulated target proteins. To identify the site of
interaction of calcium-activated calmodulin with iPLA2
, analysis of direct protein-protein interactions (protein footprinting), functional interactions (ternary complex affinity chromatography), and
biophysical interactions (fluorescence resonance energy transfer) were
pursued. Herein, we demonstrate that incubation of iPLA2
with trypsin in the presence of calmodulin and calcium ion protects against tryptic cleavage at a C-terminal site (at or near residue 630)
and increases hydrolytic cleavage near the active site of riPLA2
. Moreover, complete tryptic digestion of
riPLA2
resulted in a peptide within this 15-kDa
C-terminal region (AWSEMVGIQYFR corresponding to residues 694-705)
that binds to CaM in a calcium-dependent manner.
Collectively, these results identify the 15-kDa C-terminal region of
riPLA2
as necessary and sufficient for the
calcium-induced binding of calmodulin to riPLA2
and
conformational alteration of riPLA2
near the active site.
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EXPERIMENTAL PROCEDURES |
Materials--
The peptide corresponding to residues 23-42
(CRVKEISVADYTSHERVREEG) near the N terminus of iPLA2
(GenBank accession number I15470) was synthesized by Alpha Diagnostic
International (San Antonio, TX). The peptide corresponding to residues
731-744 (CTEVYIYEHREEFQK) near the C terminus of iPLA2
was synthesized by the Protein Chemistry Laboratory at Washington
University. Both peptides contain N-terminal cysteine residues for
covalent linkage to maleimide-activated keyhole limpet hemocyanin as a
carrier protein for generating antibodies as well as to activated
thiol-Sepharose 4B for subsequent affinity purification of the
antibodies. The synthetic 12-amino acid peptide (AWSEMVGIQYFR)
corresponding to amino acid residues 694-705 of the
iPLA2
sequence was synthesized by Research Genetics. Sequencing grade modified trypsin used for trypsinolysis of
iPLA2
was purchased from Promega. Kaleidoscope
prestained SDS-PAGE protein standards were obtained from Bio-Rad. The
µRPC C2/C18 SC 2.1/10 column, CNBr-activated Sepharose resin, and
ECL reagents were purchased from Amersham Pharmacia
Biotech.
L-
-1- Palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine,
used for measurements of riPLA2
activity, was purchased
from PerkinElmer Life Sciences. High purity bovine brain calmodulin was
purchased from Calbiochem. Calmodulin-agarose and most other reagents
were obtained from Sigma.
Generation and Affinity Purification of
Anti-iPLA2
-Antibodies--
Recombinant
calcium-independent phospholipase A2
was purified to
homogeneity from an Sf9 cell expression system by sequential affinity chromatographic steps with ATP-agarose and CaM-agarose affinity resins as described previously (21, 22). Affinity purified
polyclonal antibodies directed against riPLA2
were
prepared by initial injection of 50 µg of purified
riPLA2
in 0.5 ml of Freund's complete adjuvant and
boosted every 2 weeks with 50 µg of riPLA2
in
Freund's incomplete adjuvant until seroconversion was detected by
immunoblotting. Purified riPLA2
was coupled to CNBr-activated Sepharose according to the manufacturer's instructions to generate an affinity column to purify the anti-iPLA2
antibodies. Immunoreactive rabbit serum was loaded onto an
riPLA2
Sepharose column, and nonspecifically bound
protein was removed from the column by exhaustive washing with 10 column volumes of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl. Anti-iPLA2
antibody was eluted by
sequential application of solutions containing either 4.9 M MgCl2 or 100 mM glycine, pH 2.5. After elution,
the antibody was dialyzed against ammonium bicarbonate buffer prior to
lyophilization for storage at
20 °C.
Antibodies directed against the peptides corresponding to residues
23-42 (N-terminal) and residues 731-744 (C-terminal) regions of
iPLA2
were generated by immunizing rabbits with
conjugates of the peptides with keyhole limpet hemocyanin. Briefly,
maleimide-activated keyhole limpet hemocyanin (2 mg) was reacted with
each peptide (2 mg) according to the manufacturer's instructions and
dialyzed against 83 mM sodium phosphate, pH 7.2, containing
0.9 M NaCl. Rabbits were immunized/boosted with the
conjugates using Freund's complete/incomplete adjuvants as described
above. For affinity purification of the antibodies, each peptide (2 mg)
was covalently linked to activated thiol-Sepharose 4B (1 ml) in the
presence of an equal volume of 0.1 M sodium citrate buffer,
pH 4.5. Following an overnight incubation with mixing at room
temperature, the extent of the reaction was monitored
spectrophotometrically by the displacement of the 2-pyridyl groups as
2-thiopyridone (
343 = 8.08 × 103
M
1 cm
1). Immunoreactive rabbit
antisera were diluted 1:10 with 10 mM Tris-HCl, pH 7.5, prior to application to the appropriate affinity resin equilibrated
with the same buffer. The resins were extensively washed with 10 column
volumes of 10 mM Tris-HCl buffer (pH 7.5) containing 500 mM NaCl prior to elution of bound antibodies with 0.1 M glycine, pH 2.5, into collection tubes containing 1 M Tris-HCl, pH 9.0 (1:10 fraction volume), for
investigation. Antibodies were dialyzed against phosphate-buffered
saline and stored at 4 °C prior to use.
Partial Trypsinolysis of Purified
riPLA2
--
Purified recombinant iPLA2
(9 µg) in 25 mM imidazole, pH 8.0, containing 100 mM NaCl and 2 mM DTT was partially digested with N-tosyl-L-phenylalanine chloromethyl
ketone-modified trypsin (1:30 w/w trypsin:iPLA2
) in the
presence (1 mM CaCl2) or absence (1 mM EGTA) of calcium ion and/or CaM (9 µg, 5-fold molar
excess of CaM:iPLA2
) at room temperature. Each reaction
was terminated at the indicated time intervals (i.e. 1, 3, and 9 min) by the addition of SDS-PAGE loading bromophenol buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.025%
bromphenol blue, and 100 mM DTT). The proteolyzed
iPLA2
products were subsequently separated by SDS-PAGE
on 10% polyacrylamide gels according to the method of Laemmli (23),
transferred to polyvinylidene difluoride membranes by electroelution in
100 mM CAPS buffer, pH 11, and analyzed by ECL Western
blotting with polyclonal antibodies directed against the
iPLA2
holoprotein, the N-terminal region of
iPLA2
, or the C-terminal region of iPLA2
in conjunction with an anti-rabbit IgG horseradish peroxidase conjugate.
CaM-Agarose Affinity Chromatography of Partially Trypsinized
iPLA2
--
Purified recombinant iPLA2
(50 µg) was partially digested with trypsin (1:30 w/w
trypsin:iPLA2
) for 4-20 min at room temperature in 25 mM imidazole buffer, pH 8.0, containing 0.1 mM
EGTA, 1 mM DTT, and 50-150 mM NaCl. The
reaction was terminated by addition of 1 mM
4-(2-aminoethyl)benzene-sulfonylfluoride. Calcium chloride was added to
a final concentration of 5 mM, and the sample was applied
to a 0.5-ml CaM-agarose column equilibrated with 25 mM imidazole, pH 8.0, containing 5 mM CaCl2, 1 mM DTT, and 150 mM NaCl. The column was then
washed with equilibration buffer containing 10-fold less (0.5 mM) CaCl2, followed by elution of the bound proteins with equilibration buffer (without CaCl2)
containing 10 mM EGTA. Column fractions were analyzed by
Western analysis as described above using polyclonal antibodies
directed against the iPLA2
holoprotein.
Tryptic Digestion, Isolation, and Characterization of the
CaM-binding Peptide from riPLA2
--
A 25-µg sample
of highly purified riPLA2
was exhaustively digested with
1 µg of N-tosyl-L-phenylalanine chloromethyl
ketone-modified trypsin in 1.5 ml of 25 mM imidazole
buffer, pH 8.0, containing 1 mM EGTA for 18 h at
37 °C. The resultant proteolytic fragments were adjusted to a final
calcium ion concentration of 5 mM and loaded onto a 0.5-ml
column of CaM-agarose. The column was washed with 5 column volumes of a
buffer containing 25 mM imidazole, pH 8.0, and 0.5 mM CaCl2 prior to the elution of bound peptides with a buffer containing 25 mM imidazole, pH 8.0, and 4 mM EGTA. Aliquots of the tryptic peptides (50 µl) in the
CaM-agarose load and void fractions were directly analyzed by RP-HPLC
utilizing a µRPC C2/C18 SC 2.1/10 Smart System column with UV
absorbance detection at 215 nm. The peptides from the EGTA eluent of
the CaM-agarose column (2.0 ml) were concentrated by lyophilization, resuspended in 100 µl of deionized H2O containing 0.075%
trifluoroacetic acid (buffer A), and loaded onto a µRPC C2/C18 SC
2.1/10 RP-HPLC column. Peptides were resolved utilizing a discontinuous
linear gradient from 0 to 38% buffer B (80% acetonitrile and 0.06%
trifluoroacetic acid) for 60 min, 38 to 75% buffer B for 30 min, and
75 to 100% buffer B for 15 min. The affinity purified peptide from the
RP-HPLC purification of the calmodulin-agarose eluent (denoted in Fig. 4B) was collected, lyophilized, and sequenced by automated
Edman degradation utilizing an Applied Biosystems Procise Sequencer. RP-HPLC of the synthetic 12-amino acid peptide corresponding to the
obtained sequence (vida infra) was conducted employing the chromatographic conditions described above.
Fluorescence Spectrometry of Dansyl-CaM--
Experiments
employing dansyl-calmodulin were conducted utilizing an Aminco SLM
4800C fluorescence spectrometer (SLM Instruments, Inc.) using
established methods (24-27). Briefly, the indicated amounts of
synthetic peptide were added to a 3-ml cuvette containing 2 ml of
dansyl-CaM (1 µg/ml) in the presence of 20 mM HEPES, pH 7.2, 130 mM KCl, and 500 µM CaCl2
or 1 mM EGTA. Emission spectra were recorded from 400 to
550 nm utilizing an excitation wavelength of 340 nm.
Deletional Mutagenesis of iPLA2
--
Mutants of
iPLA2
were prepared by polymerase chain
reaction-directed mutagenesis techniques as previously described (28). Three truncated mutants of iPLA2
containing amino acid
residues 1-381, 1-600, and 1-690 were prepared. Briefly, antisense
primers containing a stop codon after amino acids 381, 600, and 690 followed by an SphI restriction site were prepared for use
in polymerase chain reaction with a sense primer containing an
EcoRI restriction site prior to the ATG start site of the
iPLA2
coding sequence. EcoRI/SalI-digested polymerase chain reaction
products were cloned into similarly digested pFAST vectors for
expression of recombinant protein in the baculovirus expression system
as previously described (22). All constructs were sequenced on both
strands to ensure fidelity of the sequence within the constructs prior
to use in the baculoviral expression system.
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RESULTS |
Previous work has demonstrated that riPLA2
reversibly binds to calmodulin through a ternary complex comprised of
calcium, calmodulin, and unidentified domains of riPLA2
(22). Accordingly, we sought to identify the specific regions of
riPLA2
involved in calmodulin binding and modulation of
iPLA2
enzymic activity. Historically, one approach
through which protein-protein contact points have been identified is
through binding of the regulatory protein to the target protein and
protection of the target protein from proteolysis at the binding site
(i.e. protein footprinting). Accordingly, we analyzed the
kinetics of the production of individual proteolytic fragments formed
during timed incubations of trypsin with riPLA2
,
calcium, and calmodulin. Incubation of trypsin with riPLA2
produced six major proteolytic fragments (A-F)
within 9 min under the conditions employed (Fig.
1). Similar kinetic analysis of tryptic
digests of riPLA2
in the presence of EGTA alone,
Ca+2 alone, or CaM and EGTA together produced similar
amounts and ratios of proteolysis products. Remarkably, tryptic digests
of riPLA2
in the presence of CaM and calcium together
resulted in the nearly complete disappearance of the major proteolytic
product at 68 kDa and the increased intensity of the band at 40 kDa
(Fig. 1, band E). These results demonstrate that
calcium-activated CaM specifically protected riPLA2
from
trypsinolysis at a single cleavage site within ~15 kDa of either its
N or C terminus. Moreover, they suggest that a site near the center of
riPLA2
(i.e. near the active site) undergoes
a conformational alteration in the presence of calcium and calmodulin
together but not with either alone. To determine the locus of the
protected site, immunoaffinity purified polyclonal antibodies directed
against either the N- or C-terminal regions of iPLA2
were generated as described under "Experimental Procedures."
Western blotting of the products from the limited trypsinolysis of
riPLA2
using the N-terminal antibody demonstrated bands
at 83 kDa (starting material) and fragments at 68, 59, and 43 kDa (Fig.
2A). In contrast, Western
blotting with the C-terminal antibody identified the 83-kDa (starting
material) as well as 53- and 40-kDa proteolyzed products (Fig.
2B). These results demonstrate that calcium-activated CaM
tightly binds iPLA2
at a specific site
15 kDa from
the C-terminal region (i.e. the production of the 68-kDa
product was dramatically attenuated in the presence of
calcium-activated CaM but not with calmodulin or calcium ion alone).
Furthermore, the results also demonstrate the conformational
modification of iPLA2
by calcium-activated CaM at a site
40 kDa from the C terminus (i.e. near the active site).

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Fig. 1.
Time course analysis of the effect of
Ca2+·CaM on limited trypsinolysis of
riPLA2 . Purified recombinant
iPLA2 (~9 µg) was incubated at 22 °C with trypsin in 25 mM imidazole buffer (pH 8.0, containing 100 mM
NaCl and 1 mM DTT) containing either Ca+2 (1 mM CaCl2), EGTA (1 mM), EGTA (1 mM) and CaM (~9 µg), or Ca+2 (1 mM CaCl2) and CaM (~9 µg). Aliquots from
each reaction were taken at the indicated times, and proteolysis was
terminated by boiling each aliquot in SDS-PAGE loading buffer for 3 min. Proteolytic products of riPLA2 (and undigested
riPLA2 ) were resolved by electrophoresis on 10%
polyacrylamide gels followed by transfer to polyvinylidene difluoride
membranes. Immunoreactive bands were detected by ECL utilizing
immunoaffinity-purified polyclonal antibodies directed against the
riPLA2 holoprotein and an anti-rabbit IgG-horseradish
peroxidase conjugate as described under "Experimental Procedures."
The first lane is riPLA2 alone incubated in buffer
containing EGTA for 9 min at 22 °C in the absence of trypsin
( T). Similar profiles were obtained in three independent
preparations. Molecular masses of marker proteins are shown on the
right in kilodaltons.
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Fig. 2.
Time course analysis of the effect of
Ca2+·CaM on limited trypsinolysis of
riPLA2 . Purified recombinant
iPLA2 (9 µg) was incubated with trypsin in 25 mM imidazole buffer, pH 8.0 (containing 100 mM
NaCl and 1 mM DTT) with either Ca+2 (1 mM CaCl2), EGTA (1 mM), EGTA (1 mM) and CaM (9 µg) or Ca+2 (1 mM) + CaM (9 µg). Aliquots from each reaction were taken at the indicated
times, boiled in SDS-PAGE buffer, electrophoresed, and transferred to
polyvinylidene difluoride membranes as described above. Immunoreactive
bands were detected by ECL utilizing immunoaffinity-purified polyclonal
antibodies directed against either the iPLA2 N terminus
(residues 23-42) (A) or the iPLA2 C terminus
(residues 731-744) (B) as described under "Experimental
Procedures." The first lane is riPLA2 alone incubated
in buffer containing EGTA for 9 min at 22 °C in the absence of
trypsin ( T). Similar results were obtained in three
independent preparations. Molecular masses of marker proteins are shown
on the right in kilodaltons.
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To substantiate the functional importance of the 15-kDa C-terminal
region of riPLA2
in the binding of calcium-activated
calmodulin to iPLA2
, ternary complex affinity
chromatography was employed. Previously, it has been established that
riPLA2
(both crude and purified) binds to CaM in the
presence of calcium ion and that it can be reversibly dissociated by
EGTA (22). Accordingly, the specific interactions engendered from
ternary complex calmodulin affinity chromatography were utilized to
discriminate between the proteolytic products of riPLA2
that retained calcium-activated CaM binding elements and those that did
not. Two approaches were employed. In the first approach, partial
tryptic digests of riPLA2
(4 min) were prepared in the
absence of calcium-activated CaM to generate (predominantly) the 68-kDa
polypeptide cleaved at or near residue 630. Following the addition of
calcium ion, the partially trypsinized mixture was loaded onto a
CaM-agarose affinity column previously equilibrated with buffer
containing 5 mM CaCl2. After washing, the
column was eluted with buffer containing 10 mM EGTA.
Western analysis of column eluates of proteolytic fragments from the
4-min digestion reaction demonstrated that the overwhelming majority
(~90%) of the 68-kDa polypeptide did not bind to the affinity
matrix, whereas >95% of the holoenzyme (83 kDa) avidly bound to the
CaM column (Fig. 3A).
Interestingly, the 40-kDa fragment (band E in Figs. 1 and
2B) corresponding to the C-terminal half of
riPLA2
containing the active site appeared to bind as
tightly to the Ca+2·CaM resin as did the 83-kDa
holoprotein (Fig. 3A). The small residual amount of binding
of the 68-kDa fragment could be due to the presence of
iPLA2
heteromultimers, which still possess at least one
intact C-terminal portion of iPLA2
(either as an intact
83-kDa component or as a noncovalently associated C-terminal fragment).
To further address this issue, riPLA2
was trypsinized for 20 min to eliminate (as much as possible) the 83-kDa holoprotein and then subjected to CaM-agarose chromatography as described above.
Under these conditions, virtually all of the 68-kDa fragment failed to
bind to the column (Fig. 3B, upper panel). In
addition, N-terminal fragments D and F were also not retained by
CaM-agarose in the presence of calcium ion (Fig. 3B,
upper panel), although untrypsinized riPLA2
bound tightly to calmodulin-agarose in control experiments (Fig.
3B, lower panel). Notably, the 68-kDa fragment retained the ability to bind to ATP-agarose, demonstrating that its
folding and structural integrity had not been significantly compromised
during limited trypsinolysis (data not shown). We specifically point
out that the present results do not exclude the possibility that some
portions of the 68-kDa N-terminal polypeptide do contact calmodulin.
However, the results do demonstrate that the C-terminal portion is both
necessary (i.e. > 90% of the 68-kDa peptide was not bound
by calmodulin) and sufficient (i.e. the 40-kDa C-terminal
peptide bound in the presence of calcium ion and was released by EGTA)
for binding of iPLA2
to calmodulin in a
calcium-dependent manner. Collectively, these results
demonstrate that the major domain responsible for riPLA2
binding to calcium-activated calmodulin is located between residues 620 and 752.

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Fig. 3.
Calmodulin ternary complex affinity
chromatography of riPLA2 tryptic
fragments. Purified recombinant iPLA2 (50 µg) was
trypsinized for 4 min (A) or 20 min (B) at
22 °C in the absence of Ca+2·CaM, after which the
reaction was terminated by the addition of 4-(2-aminoethyl)
benzene-sulfonylfluoride (1 mM). Calcium chloride was added
to a final concentration of 5 mM, and the tryptic products
were loaded on a CaM-agarose column equilibrated with 25 mM
imidazole buffer, pH 8.0, containing 1 mM DTT and 5 mM CaCl2. After washing the column with 8 column volumes of equilibration buffer containing a lower
[Ca+2] (0.5 mM CaCl2) and the
indicated concentrations of NaCl, bound protein was eluted with 10 mM EGTA in 25 mM imidazole buffer, pH 8.0, containing 1 mM DTT. Column fractions were subjected to
SDS-PAGE and analyzed by ECL immunoblot analysis using a polyclonal
antibody directed against the riPLA2 holoprotein as
described under "Experimental Procedures." Similar results were
obtained in four independent preparations.
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To identify specific portions of the 15-kDa C-terminal fragment (or
potentially other regions) that interacted with CaM in a
calcium-dependent manner, riPLA2
was
exhaustively trypsinized. SDS-PAGE of these samples demonstrated the
completeness of trypsinolysis (Fig.
4A). RP-HPLC of the
exhaustively digested riPLA2
revealed the presence of
~44 peptide peaks (Fig. 4B, Load). When
riPLA2
peptides generated by exhaustive trypsinolysis
were applied to CaM-agarose in the presence of calcium ion, a single
peptide (retention time = 62 min) was absent in the void volume
(Fig. 4B). Moreover, when peptides that bound to the CaM
matrix were subsequently eluted with buffer containing EGTA, the same
unique peptide (retention time = 62 min) was selectively
concentrated. Of the
44 peaks in the load and void fractions that
did not interact with CaM (i.e. they eluted in the void
volume), the peak at 62 min was the only peak enriched in the EGTA
elute (Fig. 4B). The amino acid sequence of the unique
calmodulin binding peptide was determined by automated Edman
degradation and identified as the 12-amino acid fragment AWSEMVGIQYFR,
which is within the previously identified 15-kDa C-terminal fragment
from calmodulin footprinting experiments (Figs. 1 and 2). This peptide
corresponds to tryptic cleavage between residues Arg693 and
Arg705.

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Fig. 4.
RP-HPLC analysis of exhaustive tryptic
digests of recombinant iPLA2 after
CaM-agarose chromatography. Peptides from the trypsin digestion of
riPLA2 were prepared and subjected to CaM-agarose
ternary complex affinity chromatography as described under
"Experimental Procedures." A illustrates the
completeness of tryptic digestion by SDS-PAGE. In B,
peptides from exhaustive trypsinolysis were applied to a CaM-agarose
affinity column in the presence of calcium ion. After washing the
column, peptides possessing an affinity for calcium-activated
calmodulin were eluted by application of buffer containing EGTA.
Peptides in each fraction were concentrated, and prepared for RP-HPLC
as described under "Experimental Procedures." RP-HPLC was performed
on an µRPC C2/C18 SC 2.1/10 RP-HPLC column, and absorbance of column
eluates was monitored at 215 nm. The large peaks from 80-95 min are
not peptides and result from concentrating uv absorbing buffer
contaminants during RP-HPLC. The peak indicated by the
asterisk represents the unique peptide derived from
riPLA2 that bound to the calmodulin-agarose resin in a
calcium-dependent manner.
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To determine whether this peptide interacted with calmodulin in a
calcium-dependent fashion, fluorescence resonance energy transfer experiments were performed. First, a peptide with this 12-amino acid sequence was synthesized and demonstrated to possess an
elution profile by RP-HPLC similar to that of the peptide
resulting from exhaustive trypsinolysis of riPLA2
(Fig.
5). Next, the 12-amino acid synthetic
peptide was incubated with dansyl-CaM and shown to interact in a
calcium-dependent manner as demonstrated by fluorescence energy transfer (Fig. 6). The increase in
fluoresence and blue shift of the emission maxima of dansyl-CaM, which
occurs only in the presence of calcium ion and the peptide, is
indicative of their close association, as has been observed with other
calcium-dependent CaM-binding peptides (24-27).

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Fig. 5.
Comparison of RP-HPLC profiles corresponding
to the peptide in the CaM-agarose eluent and the synthetic
peptide. A synthetic peptide corresponding to the amino acid
sequence of the peptide in the CaM-agarose EGTA eluent was prepared and
subjected to RP-HPLC analysis as described under "Experimental
Procedures." Trace A represents the RP-HPLC profile of the
peptide from the CaM-agarose EGTA eluent. Trace B represents
the RP-HPLC profile of the synthetic peptide.
|
|

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Fig. 6.
Fluorescence energy transfer of dansyl-CaM
with the calmodulin binding peptide. A 2-µg sample of dansyl-CaM
was incubated in the presence or absence of 60 µM
synthetic peptide (694) in 20 mM HEPES, pH 7.2, 130 mM KCl containing either 500 µM
CaCl2 (Calcium) or 1 mM EGTA. The
fluorescence intensity was measured over an emission wavelength window
from 400 to 550 nm at an excitation wavelength of 340 nm. The
arrows indicate a calcium-dependent,
peptide-mediated shift in the fluorescence intensity and emission
wavelength. The fluorescence intensity is measured in arbitrary
units.
|
|
To further substantiate the results from the partial trypsinolysis
experiments, attempts were made to generate iPLA2
deletion mutants lacking various portions of the C terminus. After
expression, these constructs were found to have barely measurable
levels of iPLA2
protein mass (<100-fold protein mass
relative to wild type). Western blot analyses demonstrated the presence
of lower molecular mass bands presumably because of rapid
proteolytic degradation of the deletion mutants lacking the C terminus
(data not shown). Accordingly, we considered the possibility that
strict conservation of the 15-kDa C-terminal region was necessary for
appropriate folding and retention of protein conformation to resist
proteolysis of the incompletely folded protein. One way to gain insight
into the importance of specific regions of primary sequence in protein structure and function is to identify highly conserved areas of amino
acid homology across species lines. Absolute complexity alignment
analysis (29) demonstrated that the CaM-binding domain was the longest
conserved region in the protein over four known species lines (Fig.
7). Thus, this region may be necessary
for the proper folding and tertiary structure of iPLA2
,
and its absence may make the protein more susceptible to Sf9
cell degradative proteases.

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Fig. 7.
Absolute complexity analysis of
iPLA2 of the four known sequences
from different species. Amino acid sequences of
iPLA2 from hamster, rat, mouse, and human were aligned
utilizing the AlignX program (Clustal W algorithm (47)) of Vector NTI
Suite 6.0 (29). From this alignment, the sum of all pairwise residue
substitution scores (using an identity score matrix) normalized by the
total number of pairs were calculated for a given 20-amino acid residue
window. A schematic of the iPLA2 sequence is presented
above with the ankyrin repeat, ATP, lipase, and putative CaM-binding
domains as indicated.
|
|
 |
DISCUSSION |
Intracellular phospholipases A2 are comprised of two
distinct families of proteins that catalyze the serine-mediated
nucleophilic attack of ester linkages in cellular phospholipids
resulting in the release of arachidonic acid from its endogenous
phospholipid storage depots. One family, the cytosolic
phospholipase A2 family, of enzymes possesses a
GXSGS sequence, whereas the other family, the
iPLA2 family, possesses a GXSTG sequence at
their active site. The iPLA2 family originated as an
evolutionary distant archetype (i.e. iPLA2
)
(30) that subsequently developed specialized domains to fulfill
specific intracellular functions. For example, iPLA2
contains eight N-terminal ankyrin repeat sequences (31) and is
modulated by calmodulin (13), whereas iPLA2
contains a
C-terminal peroxisomal localization sequence (32). The present study
demonstrates that the calmodulin-binding domain of iPLA2
is comprised of multiple contact points in the 15-kDa C-terminal
region. Calmodulin footprinting of riPLA2
with trypsin
dramatically diminished the rate of generation of the 68-kDa
riPLA2
proteolysis product (which lacks the C-terminal 15-kDa polypeptide) in the presence of calcium and CaM together but not
with either alone. This strongly suggests that calcium-activated CaM
directly binds to this region of iPLA2
, protecting it
from proteolysis. Moreover, the large majority (~90%) of the 68-kDa trypsinolysis product (lacking the 15-kDa C-terminus) failed to bind
CaM in the presence of Ca+2 (Fig. 3). Importantly, the
calmodulin binding peptide identified after exhaustive trypsinolysis is
also present within this 15-kDa fragment and directly interacts with
CaM as demonstrated by fluorescent energy transfer experiments.
Collectively, these results identify the calmodulin-binding domain in
iPLA2
as the 15-kDa C-terminal portion through both
direct physical protein-protein interactions and the
calcium-dependent functional association of this region with calmodulin.
The molecular mechanisms underlying the regulation of intracellular
phospholipases A2 has been an area of intense
investigation. Alterations in cellular calcium ion flux are an integral
part of signal transduction processes in most cell types. In the case of cytosolic phospholipase A2
, an internal C2
domain binds calcium and facilitates its translocation to specific
membrane compartments after cellular activation (33-35). In prior
studies, we have demonstrated that recombinant iPLA2
reversibly binds to CaM in the presence of calcium ion and
calcium-activated calmodulin modulates iPLA2
enzymic
activity (13). Calcium-dependent binding of CaM to
iPLA2
is an intrinsic property of this polypeptide,
which was previously exploited to obtain highly purified
riPLA2
via CaM-agarose affinity chromatography (22).
Thus, iPLA2
differs from cytosolic phospholipase A2
by requiring an exogenous protein, calmodulin, to
integrate alterations in cellular calcium ion homeostasis during
cellular activation with release of fatty acids and the generation of
lysolipids. In the present study, multiple contact points in the 15-kDa
C-terminal portion of iPLA2 have been identified (Fig.
8) as important determinants of the
calcium-dependent interaction of iPLA2
with
CaM. The demonstration of specific
Ca+2-dependent CaM footprinting during limited
trypsinolysis clearly identifies one site of
calcium-dependent binding of calmodulin at or near residue
630 in iPLA2
. Similar CaM footprinting experiments with
myosin light chain kinase (36), calcineurin A (37), and with
endothelial (38) and neuronal nitric oxide synthases (38-40) have each
provided insight into the domain structure and CaM binding sites of
these proteins.

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Fig. 8.
Amino acid sequences of the 1-9-14 and IQ
motifs of iPLA2 . The
upper sequence (residues 615-640) contains a putative
1-9-14 calmodulin-binding motif comprised of hydrophobic residues
(underlined) with interspersed positively charged amino
acids. This region is protected from trypsinolysis by calcium and
calmodulin. The lower sequence (residues 691-716) includes
the sequence of the CaM binding peptide (boxed) that
overlaps with the IQ motif (defining residues are
underlined). Positively charged residues are as
indicated.
|
|
Remarkably, addition of calcium-activated CaM to riPLA2
induced a conformational change near the active site as demonstrated by
an increase in the kinetic rate of the appearance of band E in the
presence of calcium and calmodulin together but not with either alone
(Figs. 1 and 2B). Production of band E results from hydrolysis at a site ~200 amino acids from the CaM-binding domain. The most likely reason underlying this increased rate of production of
band E is that this site interacts through space with the CaM-binding region. Accordingly, we propose a model in which the active site of
iPLA2
interacts with the CaM-binding domain (in the
absence of CaM) leading to a catalytically competent enzyme, whereas
the reversible disruption of this interaction by binding of CaM to the
15-kDa C-terminal region abrogates this interaction with resultant loss
of enzyme activity. In this model, association of calcium-activated CaM
to the C terminus of riPLA2
putatively prevents
important catalytic interactions between the CaM-binding domain and the active site. Results from site-directed mutagenesis near this region
have suggested its importance for catalytic activity of iPLA2
.2
Intriguingly, absolute complexity analysis of the four known iPLA2
sequences (human, rat, mouse, and hamster) reveals
that the amino acids residues between the 1-9-14 (vide
intra) and IQ motifs represent one of the most highly
conserved regions in the entire iPLA2
protein (Fig. 7).
Collectively, these results provide the first specific evidence of a
calcium-activated CaM-induced conformational alteration of the active
site of iPLA2
.
Proteins that interact with CaM have evolved to preserve critical
residues necessary for the recruitment and binding of calcium-activated calmodulin. Based on the analysis of calmodulin binding sites (16, 18,
19), various CaM-binding sequences have been identified that, when
modeled as an
-helix, form a predicted amphipathic structure with
hydrophobic and basic amino acid residues positioned on opposite sides
of the helix. Several calcium-dependent CaM-binding proteins (e.g. Ras-GRF (41), CDC25Mm (42), and
IRS-1 (43)) contain an IQ CaM-binding motif
(IQXXXRGXXXR) (19, 44). Notably, the 12-residue
CaM-binding peptide (AWSEMVGIQYFR) identified in this study as an
important determinant in CaM-iPLA2
interactions
possesses a shortened version of the first half of the consensus IQ
motif (I701Q702XXR705)
(Fig. 8). The apparent close proximity of the tryptophan in the 12-mer
peptide (Trp695 in iPLA2
) to
dansyl-calmodulin as demonstrated by fluorescence resonance energy
transfer further substantiates the presence of a contact point between
CaM and iPLA2
mediated by this peptide. Interestingly,
iPLA2
contains arginine residues at positions 691 and
693 that contribute to the overall positive charge of this region and
likely participate in electrostatic interactions with CaM that
contribute to the stability and high affinity binding of the complex.
Analysis of the iPLA2
sequence at the CaM
protected site shows the presence of a cluster of positively charged
residues (623RKGQGNKVKK632), one or more of
which is likely a site of trypsinolysis (Fig. 8). Extensive digestion
of iPLA2
would be expected to destroy this site, thus
explaining why it is not present in the RP-HPLC profile of the
exhaustively digested protein loaded onto the CaM affinity column
and absent in the EGTA Elute fraction (Fig. 4). Further analysis of
this region indicates a 1-8-14 pseudo-consensus sequence
(I622XXXXXXXV630XXXXI635 = 1-9-14) in which the numbers refer to the relative position of
hydrophobic residues within the sequence (19). Notably,
Ile622 and Ile635 are separated by 12 amino
acids, a distance that is believed to be critical for anchoring these
residues to the two Ca+2-binding lobes of CaM (15, 16).
Some variability has been demonstrated in the location of the central
hydrophobic residue of the 1-8-14 sequence, suggesting that the
flexible central helix of CaM is able to accommodate minor alterations
in the position of this residue (45, 46). The high net positive charge
(+5) of this putative 1-9-14 motif in iPLA2
and the
positioning of these positively charged residues (Arg623,
Lys624, Lys629, Lys631, and
Lys632) adjacent to two of the hydrophobic residues
(Ile622 and Val630) may function to stabilize
the iPLA2
·Ca2+·CaM complex through
electrostatic charge pairing with the two glutamate clusters of
calcium-activated CaM (14, 46).
In conclusion, these results illustrate: 1) the direct physical
interaction of calmodulin with riPLA2
within the 15-kDa
C-terminal region; 2) calcium-activated calmodulin-induced
conformational alterations near the iPLA2
active site;
and 3) the conservation of hydrophobic and positively charged motifs
identified in other proteins present in the calmodulin binding motif of
iPLA2
. It is hoped that identification of the domain of
riPLA2
that interacts with CaM and the demonstration of
calmodulin-induced conformational alterations of the active site will
facilitate the development of therapeutic strategies that will modulate
the activity of iPLA2
in disease processes.
 |
FOOTNOTES |
*
This work was supported jointly by Juvenile Diabetes
Foundation International File Grant 996003 and National Institutes of Health Grants 1 PO1 HL 57278-02, 2 R02 HL 41250-06A1, and
P60DK20579-22.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Washington University
School of Medicine, Div. of Bioorganic Chemistry and Molecular Pharmacology, 660 South Euclid Ave., Campus Box 8020, St. Louis, MO
63110.Tel.: 314-362-2690; Fax: 314-362-1402.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M010439200
2
D. J. Mancuso and R. W. Gross,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
iPLA2, calcium-independent phospholipase A2;
iPLA2
, calcium-independent phospholipase A2
;
riPLA2
, recombinant calcium-independent phospholipase
A2
;
BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one;
CaM, calmodulin;
DTT, dithiothreitol;
RP-HPLC, reverse-phase high
performance liquid chromatography;
PAGE, polyacrylamide gel
electrophoresis;
CAPS, 3-(cyclohexylamino)propanesulfonic acid;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
 |
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