From the Small Molecule Drug Discovery Group, Genetics Institute, Inc., Cambridge, Massachusetts 02140 and the § Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 84093
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ABSTRACT |
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The Ca2+-dependent lipid binding domain of the 85-kDa cytosolic phospholipase A2 (cPLA2) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH2-terminal fragments of cPLA2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca2+-dependent manner. Unlike other C2 domains characterized to date, the cPLA2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca2+. Binding of the cPLA2 C2 domain to vesicles in the presence of excess Ca2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA2, the cPLA2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA2 C2 domain bound to phospholipid vesicles containing sn-1 and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and-2 ester linkage are not critical for binding. These results suggest that the cPLA2 C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA2 C2 domain displayed selectivity among group IIA cations, preferring Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mM Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane.
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INTRODUCTION |
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Much of the interest in the 85-kDa cytosolic PLA2 (cPLA2)1 stems from its ability to release arachidonic acid from membranes selectively, thus initiating the biosynthesis of prostaglandins, leukotrienes, and platelet-activating factor (for review, see Ref. 1). Maximal activation of cPLA2 in intact cells requires phosphorylation by a member of the mitogen-activated protein kinase family on Ser-505 (2-5). However, in the absence of an increase in cytosolic Ca2+, even stoichiometrically phosphorylated cPLA2 fails to release arachidonic acid because Ca2+ is obligatory for binding to the membrane substrate (3-8). In cells, the increase in Ca2+ results in the selective translocation of cPLA2 to the membranes of the nuclear envelope and endoplasmic reticulum (9-11), resulting in the colocalization of cPLA2 with the downstream enzymes responsible for metabolizing arachidonic acid to prostaglandins and leukotrienes. We have shown previously that the domain responsible for the membrane association is encoded at the NH2 terminus of cPLA2 (6, 7) and serves to bring a Ca2+-independent catalytic domain to the membrane substrate in response to increases in second messenger Ca2+. This Ca2+-dependent lipid binding domain is homologous to the regulatory C2 domain originally described in the classical isoforms of protein kinase C (12) but now recognized to be present in numerous proteins, and it may serve as a paradigm to explain both the features common to these domains as well as those that provide specificity among the domains.
Homologs of the protein kinase C C2 domain have been identified in at
least four classes of eukaryotic proteins that carry out critical
functions at cellular membranes (1, 6, 13-15). These proteins include
lipid-modifying enzymes (e.g. cPLA2,
phosphoinositide-specific phospholipase C (PLC), yeast
phosphatidylserine decarboxylase-2, several isoforms of the catalytic
subunit of phosphatidylinositol 3-kinase, and a plant phospholipase D),
protein kinases (e.g. ,
,
,
,
,
, and
isoforms of protein kinase C and certain related protein kinase C),
GTPase-activating proteins (ras-GTPase-activating protein
and its relatives), and regulators of vesicle transport (e.g. synaptotagmin, rabphilin, DOC2, UNC-13, and
perforin). Although the functions of the proteins that contain C2
domains are in many cases well defined, the roles that C2 domains play
in these proteins are poorly understood. The generalized function of
the C2 domain appears to be membrane association; the ligands for C2
domains identified to date comprise various components of cellular
membranes, including phospholipids, inositol polyphosphates, and other
membrane-associated proteins (for review in detail, see Ref. 15).
Interactions between C2 domains and these ligands are predicted to
regulate the various protein-specific biochemical activities. For
example, the C2 domain of PLC-
1 has been proposed to orient or
"fix" the catalytic domain of the enzyme to the membranes after it
has been "tethered" by the binding of the pleckstrin homology
domain to phosphatidylinositol bisphosphate (16).
To study the ligand specificity of the cPLA2 C2 domain without the effects of lipid binding to the catalytic domain of cPLA2, we have expressed the cPLA2 C2 domain initially as a fusion protein and subsequently as a polypeptide refolded from inclusion bodies free of lipid contamination. This domain binds zwitterionic phospholipid vesicles composed of phosphatidylcholine preferentially over anionic vesicles in the presence of physiological levels of Ca2+. This domain is highly selective for Ca2+ over Sr2+, Ba2+, and Mg2+ with respect to vesicle binding. In addition, this domain binds to phospholipid vesicles lacking sn-1 or -2 carbonyl oxygens and is insensitive to changes in the length and degree of saturation of the sn-2 fatty acyl chain, suggesting that the specific interactions with the ligand are limited to the headgroup of the phospholipid. Based on these results, we compare the properties of the cPLA2 C2 domain with other C2 domains and other proteins that bind phospholipids in a Ca2+-dependent manner.
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EXPERIMENTAL PROCEDURES |
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DNA Constructions--
Constructions were carried out using
conventional protocols (17) to generate DNA encoding proteins that
could be expressed in mammalian COS or Chinese hamster ovary (CHO)
cells or in Escherichia coli either as fusion proteins with
glutathione S-transferase (GST) or as independent
polypeptides in inclusion bodies. DNA fragments encoding
cPLA2 residues 1-126, 1-138, 1-155, and 1-178 were
generated by polymerase chain reaction (PCR) using full-length human
cPLA2 cDNA as a template and antisense primers
incorporating a 3-stop codon and EcoRI/SalI
restriction sites. For construction of GST fusion proteins, 5
-PCR
sense primers incorporated a BglII restriction site, codons
for Gly and Ser residues as part of a thrombin cleavage site, and the
first seven codons of cPLA2. cPLA2 PCR
fragments generated from these PCRs were subcloned into the BamHI/EcoRI polylinker site of the vector
pGEX-4T-1 (Pharmacia Biotech Inc.) 3
of the GST gene. Consequently,
cPLA2 polypeptides liberated from GST fusion proteins by
cleavage with thrombin contain additional NH2-terminal
Gly-Ser residues. For construction of cPLA2 polypeptides
produced in inclusion bodies, cPLA2 cDNA that had been
modified previously by substitution of several 5
-codons with codons
preferentially utilized in bacteria served as template DNA for the PCR;
5
-PCR primers incorporated these silent changes and an
AflIII restriction site; 3
-PCR primers were the same as those used in GST fusion protein constructions. These PCR products were
subcloned into the NcoI/EcoRI sites of the vector
pTrcHisB (Invitrogen), removing the 5
-codons encoding the
NH2 terminus of the polyHis fusion protein. Consequently,
the pTrc constructs reported here utilize the trc promoter
and initiator ATG codon normally employed by His fusion proteins, yet
they encode cPLA2 residues exclusively when expressed in
E. coli. DNA was transformed into the bacterial strain
HB101. All DNA constructs were sequenced and shown to be correct.
Details of constructions may be obtained from the authors.
Protein Purification--
Bacteria expressing the appropriate
constructs were fermented to late log phase and induced with 1 mM isopropyl -D-thiogalactopyranoside at
25 °C for GST constructs and 37 °C for inclusion body proteins. For isolation of GST fusion proteins, bacterial cell pellets were lysed
by nitrogen cavitation. Supernatant was adsorbed to
glutathione-Sepharose beads (Pharmacia), and fusion proteins were
removed from beads by cleavage with thrombin (Sigma). Protein was
passed over a Mono Q column (Pharmacia) and eluted with approximately
100 mM salt. Protein was dialyzed, concentrated, and stored
in buffer (20 mM Tris (pH 7.4), and 0.1 mM
EDTA) at 4 °C. Protein was greater than 90% pure, as judged by
SDS-PAGE analysis (18). For protein expressed in inclusion bodies,
bacterial cell pellets were lysed by microfluidization. Inclusion
bodies were harvested, washed, and extracted with chloroform:methanol (2:1) to remove lipids. Inclusion bodies were solubilized in 6 M guanidine HCl (GndHCl), passed over a TSK3000 gel
filtration column (Toso-Haas) run in 8 M urea, 20 mM Tris (pH 7.4), 5 mM EDTA, and 5 mM dithiothreitol at room temperature. The appropriate peak
was passed over a Mono Q column at room temperature and eluted with
400-500 mM NaCl. Protein was refolded by diluting the
desired Mono Q peak into a solution of 0.5 M arginine HCl,
25 mM Tris (pH 8.0), and 5 mM dithiothreitol
while stirring slowly at 4 °C. Refolded protein was diluted slowly
2-fold into buffer (25 mM Tris (pH 8.0) and 5 mM dithiothreitol) and again 2-fold into a solution of 2 M (NH4)2SO4, 20 mM Tris (pH 8.0), 5 mM dithiothreitol, and 5 mM EDTA. Refolded protein was adsorbed to a Toyo-phenyl 650S column (Toso-Haas) equilibrated with buffer containing 1 M (NH4)2SO4 and eluted
in a single step with salt-free buffer. Eluted protein was concentrated
by vacuum dialysis and passed over a Mono Q column. Correctly folded
protein eluted with approximately 100 mM NaCl, whereas
incorrectly folded protein, as judged by fluorescence spectroscopy (see
below), eluted with 400-500 mM NaCl. Correctly folded
protein was passed over a phenyl-5PW column (Toso-Haas) and eluted with
a linear gradient of decreasing
(NH4)2SO4. Correctly folded protein
eluted with approximately 100 mM
(NH4)2SO4, whereas incorrectly
folded protein eluted during the final column wash in salt-free buffer.
Protein was concentrated by vacuum dialysis and dialyzed against
storage buffer. Protein purity was evaluated by SDS-PAGE on 4-20% and
14% Tris-glycine and 10% Tricine gels (18).
Natural Membrane Binding Assay-- The Ca2+-dependent binding of recombinant proteins to natural membranes isolated from CHO cells was determined as described in detail previously (6, 7). In short, at least 2 µg of test protein was mixed with CHO cell membranes in the presence of 150 mM NaCl, 20 mM HEPES (pH 7.4), and Ca2+/EGTA buffers to maintain the desired free Ca2+ concentration and incubated for 15 min at 30 °C. After centrifugation at 100,000 × g, equal proportions of supernatant and pellet fractions were subjected to SDS-PAGE on 4-20% Tris-glycine gels. Gels were blotted onto nitrocellulose, and test proteins appearing in supernatant and pellet fractions were detected by immunostaining with cPLA2 antisera and visualized by chemiluminescence.
Synthetic Phospholipid Binding Assays--
The binding of the
cPLA2 C2 domain, which acts as the fluorescence donor, to
small unilamellar phospholipid vesicles containing the fluorescent
probe dansylphosphatidylethanolamine (dansyl-PE) (Molecular Probes,
Eugene, OR), the fluorescence acceptor, was measured as described
previously (7) with modification. A total of 25-50 µg of test
protein was diluted into a 2-ml solution containing 60 µg of test
liposomes (composed of 5-10% dansyl-PE) in 150 mM NaCl,
20 mM HEPES (pH 7.4), and 1 mM EGTA in 3-ml
quartz cuvettes. In experiments comparing different divalent cations,
binding reactions were carried out in buffer containing 100 mM KCl, 20 mM HEPES (pH 7.4), and 1 mM EDTA. KCl was diluted from a Ca2+-free
Ca2+ electrode-filling solution (Orion Research
Incorporated, Boston). Reaction mixtures were stirred continuously,
maintained at 20 °C, and illuminated with 284 nm wavelength light;
emission of dansyl-PE was recorded at a wavelength of 520 nm. Levels of
free divalent cations were raised by the addition of concentrated
stocks of cations: for most experiments, a Ca2+ atomic
absorption standard (VHG Laboratories, Manchester, NH) was used. Free
Ca2+ levels were calculated using the Chelator program (19)
taking into account the pH of the binding reactions, measured
separately. For experiments testing different divalent cations, the
chloride salt of the metal ions (Fluka, puriss grade) was used; free
cation levels were calculated according to Raaflaub (20). The
fluorescence emission of dansyl-PE in the presence of EGTA or EDTA
(I0) was subtracted from that in the presence of
added metal ion (I) to determine energy transfer induced by
the added metal ion, which was normalized to give (I I0)/I0, expressed as a
percentage. Vesicle compositions were varied according to the
experiment. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(PO-PC), -phosphoethanolamine (PO-PE), -phosphate (PO-PA),
-phosphoserine (PO-PS) and -phospho-rac-(1-glycerol) (PO-PG),
L-
-phosphatidylinositol (mixed acyl-PI),
1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PP-PC),
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PA-PC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine
(PL-PC), 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (as
sn-1 and -2 ether-linked phospholipid) and brain
sphingomyelin were purchased from Avanti Polar Lipids (Alabaster,
AL).
Fluorescence Spectroscopy-- A total of 2 µg of the C2 domain, diluted into a 1-ml solution, with or without denaturant, containing 150 mM NaCl, 20 mM Tris (pH 7.4), and 5 mM EDTA, was excited with 280 nm wavelength light in a 1.5-ml quartz cuvette at room temperature. Fluorescence emission wavelengths were scanned from 300 to 400 nm. Excitation and emission slit widths were both 5 nm. Similar emission maxima (but of lower intensity) were recorded when 294 nm wavelength excitation light was used. For renaturation experiments, a concentrated stock solution of protein prepared in a high concentration of denaturant was serially diluted into buffer without denaturant.
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RESULTS |
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The NH2-terminal 178 residues of cPLA2
(cPLA2(1-178)) contain a C2 domain, a sequence motif that
functions as a Ca2+-dependent lipid binding
domain (6, 7). Previously, cPLA2(1-178) was expressed as
an independent polypeptide in COS cells and bound natural membranes
in vitro at Ca2+ concentrations that activated
enzymatic activity of cPLA2 (7). In addition, these
residues conferred upon a heterologous fusion protein the ability to
bind to natural membranes and synthetic phospholipid vesicles in the
presence of physiological levels of Ca2+ in
vitro (7). We sought to define better the minimal residues of the
C2 domain. Based on the structure of the first C2 domain of
synaptotagmin (21, 22), the cPLA2 C2 domain was predicted to terminate prior to the exon boundary at Gln-126 (Fig.
1). However, sequence alignment based on
the report of a second C2 domain topological fold, that of PLC-1
(16), in which the
-strand that corresponds topologically to the
first
-strand of synaptotagmin is located at the COOH terminus of
the PLC-
1 C2 domain, predicted that the cPLA2 C2 domain
terminates at the next exon boundary Val-138 (15). Thus,
cPLA2 was COOH-terminally truncated at Gln-126 and Val-138, two exon boundaries, to ascertain whether they demarcated minimal functional boundaries. cPLA2 was also truncated in a polar
stretch of the polypeptide at Lys-155 to test the importance of
residues between 138 and 178. These polypeptides were expressed in COS or CHO cells as independently expressed polypeptides, or they were
expressed in E. coli as fusion partners of GST or as
inclusion body proteins that could be refolded.
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Recombinant fragments containing, at a minimum, residues 1-155, were expressed in both COS and CHO cells as soluble polypeptides that bound cellular membranes in a Ca2+-dependent manner (data not shown). In contrast, both cPLA2(1-126) and (1-138) were expressed as insoluble polypeptides in COS cells. We turned to expression of recombinant protein in E. coli, reasoning that expression of cPLA2(1-126) and (1-138) in bacteria grown at lower temperatures in fusion to a highly soluble partner (GST) might overcome the solubility problems associated with protein overexpression in COS cells cultured at 37 °C. Indeed, recombinant fragments containing, at a minimum, residues 1-126 attached to the COOH terminus of GST were expressed as soluble fusion proteins in E. coli. However, GST-cPLA2(1-126) was considerably more insoluble than the other three fusion proteins; the little soluble cPLA2(1-126) that could be liberated from GST-cPLA2(1-126) bound in a Ca2+-independent manner to cellular membranes (data not shown). GST-cPLA2(1-138), (1-155), and (1-178) were fully functional, binding in a Ca2+-dependent manner in vitro to cellular membranes (data not shown). When liberated from GST-fusion proteins by thrombin cleavage and purified, cPLA2(1-138) and (1-155) reversibly bound to phosphatidylcholine vesicles (Fig. 2) and cellular membranes (data not shown) at low micromolar Ca2+ levels. The additional Gly-Ser residues attached to the NH2 terminus of cPLA2(1-138) and (1-155) to create a thrombin cleavage site (see "Experimental Procedures") thus do not hamper binding to phospholipid vesicles. Although cPLA2(1-138) represented the minimal, fully functional C2 domain, we chose to characterize cPLA2(1-155) initially because of its higher yields.
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cPLA2(1-155) contains several aromatic residues, including
a single tryptophan, Trp-71, which serves as a useful spectroscopic probe. Excitation of cPLA2(1-155) at 280 nm resulted in a
strong fluorescence emission wavelength maximum at approximately 325 nm
(Fig. 3A), which is
characteristic of tryptophan buried in a relatively nonpolar
environment (23). When diluted into high concentrations of urea or
GndHCl, this fluorescence wavelength maximum red-shifted to
approximately 348 nm, characteristic of tryptophan exposure to polar
solvent. This indicates that Trp-71 of native cPLA2 is
buried in a relatively nonpolar environment and that upon denaturation
it becomes exposed to solvent. The spectroscopic properties of Trp-71
in cPLA2 are consistent with the location of the homologous
residues Phe-206 of synaptotagmin I (21) and Trp-684 of PLC-1 (16),
which are buried in the
5- and
4-strands of type I and II C2
domains, respectively (see Fig. 1). Titration of
cPLA2(1-155) with urea or GndHCl revealed a single major
transition in unfolding (Fig. 3B). When
cPLA2(1-155) was first denatured in urea or GndHCl and
then serially diluted into buffer without denaturant, the fluorescence
wavelength maximum blue-shifted back to approximately 325 nm (Fig.
3C), revealing that denaturation by these chaotrophic agents
is reversible. Further, cPLA2(1-155), which first had been
subjected to urea or GndHCl concentrations that denatured the protein
then was refolded by 100-fold dilution into buffer without denaturant,
exhibited nearly the same Ca2+ dependence for binding to
phosphatidylcholine vesicles as native cPLA2(1-155) (Fig.
4). However, it appeared that
denaturation and renaturation under these conditions reduced the yield
of the protein (see Fig 4. inset).
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Given the ability of cPLA2(1-155) derived from GST fusion protein to refold into functional protein, cPLA2(1-126), (1-138), (1-155), and (1-178) were expressed in E. coli inclusion bodies, extracted with a chloroform and methanol mixture (2:1) to remove endogenous lipids, purified, and refolded. Partially purified, refolded cPLA2(1-126) displayed a fluorescence maximum near 331 nm and failed to bind vesicles in response to micromolar levels of Ca2+ (data not shown), suggesting that it had either refolded improperly or that a critical functional portion of the molecule was missing. In contrast, purified refolded cPLA2(1-138) displayed a fluorescence maximum near 325 nm (data not shown), suggesting that cPLA2(1-138) had refolded into a native conformation. cPLA2(1-138) refolded from inclusion bodies bound to natural membranes in a Ca2+-dependent manner (data not shown) and reversibly bound phosphatidylcholine vesicles in a Ca2+-dependent manner (Fig. 5A). The phospholipid binding characteristics of lipid-free preparations of cPLA2(1-138) were characterized in greater detail.
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To ascertain if the cPLA2 C2 domain interacts directly with the phospholipid headgroup, the binding of the cPLA2 C2 domain to a variety of synthetic vesicles was measured. Reversible binding of cPLA2(1-138) to vesicles containing phosphatidylethanolamine followed the same Ca2+ dependence as binding to phosphatidylcholine; however, the maximal Ca2+-induced fluorescence transfer observed (approximately 10%) was much lower than that observed for phosphatidylcholine (approximately 80%). Because the maximal signal observed for phosphatidylethanolamine could not be increased by increasing the concentration of vesicles (data not shown), the lower maximal signal observed for phosphatidylethanolamine vesicles was likely the result of a lower efficiency of fluorescence resonance energy transfer rather than a lower affinity of C2 domain binding. Differences in the ability of dansyl-PE to pack into the two types of vesicles might account for differences in their ability to accept fluorescence energy from bound proteins. cPLA2(1-138) also bound to vesicles containing phosphatidic acid (Fig. 5B); however, approximately 10-fold greater free Ca2+ was required for half-maximal binding to these vesicles compared with phosphatidylcholine and phosphatidylethanolamine. In addition, because not all of the binding to vesicles containing phosphatidic acid was reversed by EGTA treatment (see legend to Fig. 5), a fraction of the binding observed to these vesicles was Ca2+-independent. No significant Ca2+-dependent binding of cPLA2(1-138) to vesicles containing phosphatidylserine, phosphatidylglycerol, or phosphatidylinositol was observed (Fig. 5B), even at higher vesicle concentrations (data not shown). Thus, the cPLA2 C2 domain displays dramatic selectivity for zwitterionic phospholipid headgroups in the presence of physiological levels of Ca2+.
The phospholipid headgroup selectivity of the cPLA2 C2 domain was confirmed further using a direct binding assay (Fig. 6). In this assay, the cPLA2 C2 domain complexed to phospholipid vesicles was isolated by centrifugation, subjected to SDS-PAGE, and then quantified by staining with Coomassie Brilliant Blue and densitometry. Synthetic vesicles composed of phosphatidylcholine, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol were tested for their ability to bind to the C2 domain in the presence or absence of Ca2+. We could not test phosphatidylethanolamine vesicles in this manner because PO-PE vesicles could not be collected in comparable yields by centrifugation (see "Experimental Procedures"). As observed by fluorescence resonance energy transfer, the cPLA2 C2 domain bound preferentially to phosphatidylcholine vesicles in a Ca2+-dependent manner. In addition, Ca2+-independent binding to phosphatidic acid vesicles and little or no Ca2+-dependent binding to vesicles composed of phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol were observed.
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Because molar concentrations of neutral salts have been shown to promote cPLA2 activity in vitro (24, 25), the ability of several salts to promote vesicle binding by the cPLA2 C2 domain was investigated. High concentrations of Na2SO4 promoted reversible Ca2+-independent binding of cPLA2(1-138) to vesicles containing phosphatidylcholine (Fig. 7). Half-maximal binding was observed above 1 M Na2SO4. High concentrations of (NH4)2SO4 (approximately 2 M) and NaCl (above 3 M) also promoted binding. At greater than 4 M NH4Cl no binding of cPLA2(1-138) was observed. Ca2+-independent binding of the cPLA2 C2 domain to vesicles elicited by these salts did not depend as dramatically on the identity of headgroup of the phospholipid, in contrast to Ca2+-dependent binding (Figs. 5 and 6), since we observed salt-induced binding to vesicles containing phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol (data not shown). These results demonstrate that phospholipid vesicles containing phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol are capable of acting as fluorescence acceptors for cPLA2(1-138) and serve as controls for the Ca2+-induced vesicle binding assay described above. However, vesicles containing phosphatidylcholine displayed significantly better binding at lower salt concentrations and greater efficiency of fluorescence energy transfer at saturating salt concentrations compared with vesicles composed of these other phospholipids.
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Since cPLA2 cleaves the unsaturated arachidonoyl sn-2 chain (20:4) at least 20-fold more efficiently than the shorter saturated palmitoyl chain (16:0) (6, 26, 27), the dependence of C2 domain activity on length and degree of saturation of the fatty acyl chain was tested. In contrast to the headgroup selectivity noted above, cPLA2(1-138) reversibly bound to phosphatidylcholine vesicles containing sn-2 oleoyl (18:1), linoleoyl (18:2), arachidonoyl (20:4), and palmitoyl (16:0) chains, exhibiting similar Ca2+ dependences (Fig. 8). The weaker maximal fluorescence signals obtained for PA-PC and PP-PC were likely caused by a lower efficiency of energy transfer rather than a lower affinity for binding, since an increase in vesicle concentration had no effect (data not shown). Therefore, the cPLA2 C2 domain does not display selectivity for the structure of the sn-2 acyl chain in Ca2+-dependent binding to phosphatidylcholine.
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Given that the ternary Ca2+-phospholipid-protein complex observed in crystal structures of the low molecular weight secreted by PLA2 show a direct interaction between the sn-2 carbonyl and Ca2+ (28, 29), the effect of altering the linkages between the glycerol backbone and the fatty acyl chain was probed. cPLA2(1-138) reversibly bound in a Ca2+-dependent manner to vesicles composed of brain sphingomyelin (Fig. 9), which contains an amide linkage at the sn-2-like position and a free hydroxyl at the sn-1 position. cPLA2(1-138) also bound reversibly in a Ca2+-dependent manner to vesicles composed of phospholipids containing ether linkages in place of ester linkages at the sn-1 and -2 position (Fig. 9). The same Ca2+ dependence was observed for the binding of cPLA2(1-138) to vesicles composed of sphingomyelin or glycerophospholipids bearing ester or ether linkages; however, the maximal signal obtained for sphingomyelin was not as great as that for the ester- or ether-linked phospholipids, possibly because of lower energy transfer efficiency. Thus, the sn-1 and sn-2 carbonyl oxygens are not critical for Ca2+-dependent binding of the cPLA2 C2 domain to phospholipid vesicles.
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To ascertain the cation selectivity of the cPLA2 C2 domain, vesicle binding was tested in the presence of various divalent metals. cPLA2(1-138) bound to phosphatidylcholine vesicles in the presence of Ca2+, Sr2+, and Ba2+; this binding was reversible upon cation chelation (Fig. 10). However, approximately 50-fold greater concentration of Sr2+ and nearly 10,000-fold greater Ba2+ over Ca2+ was required for half-maximal binding (Fig. 10). No significant binding to vesicles was observed in the presence of greater than 10 mM Mg2+. Therefore, the cPLA2 C2 domain displays at least 10,000-fold selectivity for Ca2+ over the other physiological cation Mg2+ in binding to phosphatidylcholine.
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DISCUSSION |
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In this report we characterize cPLA2(1-138) as a
minimal NH2-terminal fragment of cPLA2 that
functions as a Ca2+-dependent lipid binding
domain. Interestingly, Val-138 corresponds to the end of a short exon
that begins with Val-127 (1, 6). Shortening this domain to the next
nearest COOH-terminal exon boundary (after Gln-126) produced a
polypeptide that is likely to be folded improperly when expressed in
E. coli for the following reasons: (a) a greater
fraction of GST-cPLA2(1-126) was expressed as insoluble
material in E. coli compared with other GST-C2 domain fusion
proteins; (b) when liberated from the limited GST fusion protein that could be isolated, cPLA2(1-126) bound CHO
cell membranes in a Ca2+-independent manner; (c)
cPLA2(1-126) refolded from inclusion bodies failed to bind
to vesicles in response to Ca2+ or high concentrations of
NA2SO4 (data not shown). These results support
the hypothesis that the cPLA2 C2 domain forms the
alternative type II C2 domain topology represented by PLC-1 (16,
30), which was predicted by sequence alignment (15).
The metal-dependent phospholipid binding of the
cPLA2 C2 domain displays selectivity among group IIA
cations in the order of preference Ca2+ > Sr2+ > Ba2+ > Mg2+. Notably, the same preference
was observed for hydrolysis of phospholipid vesicles by the full-length
cPLA2 (24, 31). The pattern of metal-selective vesicle
binding by the cPLA2 C2 domain is identical to that
observed for the binding of the first C2 domains of synaptotagmin to
phosphatidylserine vesicles (32, 33). The cation selectivity of
rabphilin has not been reported in detail; however, under the assay
conditions reported, Ca2+, but not Ba2+,
Sr2+, or Mg2+, promoted phospholipid binding
(34). Because Ca2+ is the sole metal capable of promoting
membrane binding at physiological concentrations, these C2 domains must
possess metal ion binding sites optimized for divalent ions the size of
Ca2+, as has been observed in other Ca2+
binding sites that exclude Mg2+ (35). As observed for
phospholipid binding by the synaptotagmin C2 domain (32, 33, 36),
vesicle binding by the cPLA2 C2 domain induced by
Ca2+ is positively cooperative, suggesting the presence of
multiple Ca2+ ions in the
phospholipid·Ca2+·C2 domain ternary complex. This
finding is consistent with NMR and crystallographic studies of the C2
domains of synaptotagmin and PLC-1, which support the presence of
multiple Ca2+ binding sites (16, 20, 21, 30).
The phospholipid preference of the cPLA2 C2 domain is
distinct from that described for isolated C2 domains of other proteins characterized to date. Whereas C2 domains of most synaptotagmins, rabphilin, DOC2 (and full-length protein kinase C-) require the presence of anionic but not zwitterionic phospholipid in vesicles for
binding (7, 32, 34, 36-38), at physiological ionic strength and
Ca2+, the cPLA2 C2 domain binds preferentially
to vesicles comprised of phosphatidylcholine. It is worth noting that
the residues separating the aspartates in the second Ca2+
binding loop (the loop between strands
6 and
7 in type I C2 domains) of synaptotagmin, rabphilin, DOC2, and protein kinase C-
contain positively charged residues, whereas the corresponding short
stretch in cPLA2 (the loop between strands
5 and
6 in type II C2 domains) contains hydrophobic residues (see Fig. 1). In
addition, in place of the histidine immediately preceding the last
Ca2+ coordinating residue in synaptotagmin, the C2 domain
of cPLA2 contains an aspartate. Perhaps the choline
headgroup interacts with these acidic and hydrophobic residues in this
loop of cPLA2 but is repelled by the basic residues of C2
domains which interact with anionic vesicles. Interestingly, the C2
domain of cPLA2 binds to vesicles containing the small
anionic headgroup phosphate of phosphatidic acid to some extent at high
Ca2+ concentrations.
To date, little information has been published addressing the critical interactions between the phospholipid and C2 domains. Because we were able to refold the cPLA2 C2 domain from inclusion bodies, we had the opportunity to extract first any endogenous lipids that might have compromised our studies. The preference of the cPLA2 C2 domain for the neutral headgroup phosphocholine contrasts sharply with the headgroup preference at the active site of cPLA2 where less than 4-fold selectivity has been observed among phosphocholine, phosphoethanolamine, phosphate, phosphoserine, and phosphoinositol, despite their large structural differences (26, 27). Because phosphatidylmethanol was used to trap cPLA2 in these experiments, it is noteworthy that we have observed Ca2+-dependent binding of the cPLA2 C2 domain to vesicles containing this nonphysiological headgroup (data not shown).
The lack of selectivity for the sn-2 acyl chain in vesicle binding by the cPLA2 C2 domain is strikingly different from the selectivity observed in liposome hydrolysis by the full-length enzyme. The cPLA2 C2 domain does not distinguish among acyl chains in the sn-2 position, whereas the catalytic center prefers the unsaturated arachidonoyl (20:4) chain by 20-40-fold over the saturated palmitoyl (18:0) chain (6, 26, 27, 39-43).
We have also demonstrated that vesicle binding by the cPLA2 C2 domain does not require the carbonyl oxygens of the ester linkage at either the sn-1 or -2 positions by showing equivalent binding to either natural diacylphosphatidylcholine or synthetic phosphatidylcholine containing ether linkages at the sn-1 and -2 positions. We also showed that the cPLA2 C2 domain binds to sphingomyelin, which contains an amide linkage at the sn-2-like position. Together these results indicate that the C2 domain does not coordinate Ca2+ via the sn-2 carbonyl oxygen. Similarly, acyl linkages are not required for the binding of annexin V to phospholipid vesicles in the presence of Ca2+; in the crystal structure of annexin V bound to Ca2+ and a phospholipid analog, Ca2+ was coordinated via an sn-3 phosphoryl oxygen (44). In contrast, the sn-2 carbonyl oxygen (in conjunction with an sn-3 phosphoryl oxygen) coordinates Ca2+ when bound to the structurally and mechanistically dissimilar secreted PLA2 (28, 29).
Our results indicate that hydrolysis of phospholipid vesicles by
cPLA2 induced by high salt concentrations in the absence of
Ca2+ as observed previously (24, 25) is likely due to
salt-induced Ca2+-independent phospholipid binding by its
C2 domain and Ca2+-independent hydrolysis of the substrate.
We observed previously that a recombinant cPLA2 lacking the
C2 domain failed to hydrolyze phospholipid vesicles in the presence of
high concentrations of salt, even though this protein hydrolyzed
monomeric substrates at wild type rates in the absence of
Ca2+ (7). We show here that the C2 domain of
cPLA2 binds to phospholipid vesicles in the absence of
Ca2+ when subjected to high concentrations of select salts.
The extreme steepness of the binding curves as a function of salt
concentration and potency in the molar range suggest that salt-induced
phospholipid binding results from the effects of multiple ionic
interactions with the protein or solvent (or vesicle) rather than
substitution of a cation for Ca2+ in the metal binding
site. These salts are likely to stabilize a hydrophobic interface of
the C2 domain because the relative potency of these ions
(Na+ > NH4+;
SO42 > Cl
) follows
exactly the Hofmeister series of cations and anions that promote
hydrophobic interactions (45). This hydrophobic interface may also be
stabilized by Ca2+ binding to the C2 domain and might
interact directly with a hydrophobic portion of the phospholipid
vesicle or form an allosteric switch to induce vesicle binding
elsewhere in the C2 domain. C2 domains that bind phospholipids in a
Ca2+-independent manner may have evolved to stabilize this
interface constitutively.
Our experiments have not addressed the reason why full-length cPLA2 translocates selectively to the nuclear and endoplasmic reticulum membranes over plasma membranes (9-11, 46) or even if this preference is encoded within the C2 domain. The preference may be caused by interaction of cPLA2 with a docking protein localized to the these membranes, or it may be the result of differences in the composition of membranes. For example, cPLA2 may translocate to these membranes selectively over the plasma membrane because the latter contains high levels of sphingomyelin and cholesterol, both of which promote order and tight packing of phospholipids. Nevertheless, the cPLA2 C2 domain was shown to bind with high affinity to the vesicles composed of sphingomyelin. Similarly, in experiments not shown, we have observed that high levels of cholesterol also do not block binding to the membrane. Interestingly, sphingomyelin has been reported to inhibit a partially purified cPLA2 (47). This result, together with the finding that full-length cPLA2 binds more tightly to membranes containing products of phospholipid hydrolysis (31), suggests that sphingomyelin content may influence binding, not by blocking the initial binding event but by inhibiting the PLA2 reaction that promotes increased binding.
In conclusion, we have demonstrated that the first 138 amino acids of
cPLA2, ending at an exon border, constitute a minimal NH2-terminal fragment of cPLA2 which contains a
fully active C2 domain. This finding suggests that the
cPLA2 C2 domain possesses a topology similar to the C2
domain of PLC-1 rather than that of the first synaptotagmin C2
domain. By observing a selectivity among headgoups and yet no
preference in the fatty acyl chains or the linkages to the glycerol
backbone, we have also determined the portions of the phospholipid
which are likely to intimately contact the C2 domain. Finally, we have
demonstrated that Ca2+ is the only divalent metal that
promotes binding at physiological concentrations, thus reinforcing the
concept that the cPLA2 C2 domain and the
Ca2+-dependent C2 domains in general link
second messenger Ca2+ to protein function.
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ACKNOWLEDGEMENTS |
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We thank Dale Cumming for advice, Neil Schauer for large scale bacterial fermentation, Elliott Nickbarg for mass spectroscopic analysis, and John Knopf for continuing support and advice.
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FOOTNOTES |
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* 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.
Supported by National Institutes of Health Postdoctoral Fellowship
GM 18303. Present address: Dept. of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 84093.
¶ Supported by National Institutes of Health Grant 48203.
To whom correspondence should be addressed: Small Molecule
Drug Discovery Group, Genetics Institute, Inc., 87 Cambridge Park Dr.,
Cambridge, MA 02140. Tel.: 617-498-8944; Fax: 617-498-8993.
1 The abbreviations used are: cPLA2, cytosolic phospholipase A2; C2, second conserved; PLC, phospholipase C; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GndHCl, guanidine hydrochloride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; dansyl-PE, N-(5-dimethylaminonaphthalene-1-sulfonyl)-1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine; PO-PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PO-PE, -phosphoethanolamine; PO-PA, -phosphate; PO-PS, -phosphoserine; -PG, phospho-rac-(1-glycerol); mixed acyl-PI, phosphatidylinositol containing a mixture of acyl chains; PP-PC, 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine; PA-PC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PL-PC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine; sn-1 and -2 ether-linked phospholipid, 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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