(Received for publication, October 16, 1996, and in revised form, January 15, 1997)
From the Department of Chemistry and Biochemistry,
University of California at San Diego,
La Jolla, California 92093-0601 and the § Genetics
Institute, Cambridge, Massachusetts 02140
A novel Ca2+-independent phospholipase A2 (iPLA2) has recently been purified and characterized from P388D1 macrophages (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions. Also an iPLA2 from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575). We report herein that the cloned CHO iPLA2 is equivalent to the mouse enzyme purified from P388D1 cells. Polymerase chain reaction amplification of cDNA fragments from P388D1 cells using primers based on the CHO iPLA2 sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D1 cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions. Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D1 enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate. Additionally, covalent modification of the CHO enzyme by [3H]bromoenol lactone is dependent on active enzyme as is the P388D1 iPLA2. Finally, both enzymes have the same specific activities under identical experimental conditions.
Phospholipase A2 (PLA2)1 comprises a superfamily of enzymes that regulate phospholipid metabolism and generate bioactive lipid mediators such as the eicosanoids and platelet-activating factor (for review see Ref. 1). Since the PLA2s have been implicated in a number of tissue dysfunctions ranging from inflammation to ischemia, much attention has been devoted to the study of the mechanism of action and biochemical characteristics of these enzymes. Depending on their site of action, the PLA2s can be subdivided into two types: the extracellular, secreted enzymes and the intracellular, cytoplasmic enzymes (1). Among the latter, two groups of enzymes can be considered, namely the group IV, Ca2+-dependent cytosolic PLA2, and the Ca2+-independent PLA2 (iPLA2).
In contrast to the group I-IV PLA2s, the Ca2+-independent PLA2s (iPLA2) have been poorly studied, since most of them are labile, constitute only a minor fraction of the total cellular protein, and have lower specific activities. The iPLA2s have been grouped into three main categories based on their biochemical and localization characteristics: lysosomal, brush-border membranes, and intracellular (for review, see Ref. 2). The lysosomal and brush-border membrane iPLA2s appear to be conserved among the species where they have been identified. However, the intracellular iPLA2 enzymes appear to represent a much more diverse and broad group of enzymes, whose relationship is not so evident. Only four intracellular iPLA2s have been purified to homogeneity, namely a 40-kDa enzyme from myocardium (3), a 39-kDa enzyme from bovine brain (4), an 80-kDa enzyme from P388D1 macrophages (5), and very recently a 28-kDa enzyme from rabbit kidney (6). The enzymes from myocardium and P388D1 cells are both modulated by ATP and form oligomeric complexes of about 400 kDa. However, besides their very distinct molecular mass, they also differ significantly in substrate preference and detergent sensitivity (3, 5).
The first molecular cloning of an iPLA2 from CHO cells is reported in the accompanying manuscript (7). Due to the role of iPLA2 in P388D1 cell metabolism (8), it was essential to determine whether or not the enzyme present in these cells was the same as that cloned by Tang et al. (7). Using a variety of techniques, we demonstrate herein that the enzyme from P388D1 cells and the cloned enzyme from CHO cells is the same molecular entity expressed in different species. Thus, the availability of the macrophage iPLA2 sequence should ensure rapid progress in understanding its physiological function.
1-Palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine was obtained from Amersham Corp. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine was purchased from Avanti (Birmingham, AL). (E)-6-(bromomethylene)-tetrahydro-3-(1-[4-3H]naphthalenyl)-2H-pyran-2-one ([3H]BEL) was a generous gift of Drs. Randy H. Weiss and Philip Needleman (Monsanto Co., St. Louis, MO). Palmitoyl trifluoromethyl ketone (PACOCF3) and BEL were synthesized in our laboratory as described previously (9). Methyl arachidonyl fluorophosphonate (MAFP) was purchased from Cayman Chemical Co. (Ann Arbor, MI).
iPLA2 AssayThe iPLA2 activity has
been previously described (5). Briefly, 10-50 ng of purified
iPLA2 was assayed in a buffer consisting of 100 mM Hepes, 400 µM Triton X-100, 100 µM
1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine
(200,000 cpm), 5 mM EDTA, and 0.1 mM ATP (pH
7.5). The mixture was incubated at 40 °C for 30 min with shaking,
the reaction was stopped by adding 2.5 ml of Dole reagent
(2-propanol, heptane, 0.5 M H2SO4;
400:100:20 (v/v/v)), and products were processed by a modified Dole
extraction procedure (10). When the irreversible inhibitor BEL, MAFP,
or 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was used in the assays, the enzyme was preincubated with the inhibitor for 5 min at 40 °C. The remainder of enzyme activity was assayed by adding the substrate mixture. When the reversible inhibitor PACOCF3 was used, it
was added directly to the assay mixture.
Purified proteins were analyzed by a 10% SDS-polyacrylamide gel electrophoresis (Novex), transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp.). Nonspecific binding was blocked by incubating the membranes with a buffer consisting of 5% nonfat milk, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 for 60 min. Membranes were incubated with the antiserum generated against the CHO iPLA2 (1:200 into the blocking buffer) for 30 min and then treated with horseradish peroxidase-conjugated protein A (Amersham). Bands were detected by enhanced chemiluminiscence (ECL; Amersham).
[3H]BEL Labeling100 ng of the CHO or P388D1 cell iPLA2 was incubated with 7 µM [3H]BEL for 30 min at 40 °C. Excess unreacted [3H]BEL was washed out eight times with H2O using a Centricon 10 (Amicon Inc.). Samples were then analyzed by 10% SDS-polyacrylamide gel electrophoresis (Novex). Gels were fixed with 40% methanol, 10% acetic acid and incubated for 30 min with a fluorographic reagent (Amplify, Amersham). Bands were visualized by autoradiography (9). When MAFP was used to prevent [3H]BEL binding, purified enzyme was preincubated with 20 µM MAFP for 15 min at 40 °C prior to the addition of [3H]BEL.
P388D1 iPLA2 SequencingTotal RNA
from P388D1 cells was isolated using the guanidinium
isothiocyanate/phenol chloroform extraction method (Stratagene). Polyadenylated mRNA was purified by affinity chromatography using an oligo(dT)-cellulose column (Stratagene). The mRNA was converted to cDNA using the Stratagene reverse transcriptase-PCR kit.
Amplification of specific cDNA fragments was achieved by using
recombinant Pfu polymerase (Stratagene). Primers were based
on the CHO iPLA2 sequence (Ref. 7; GenBankTM
accession number I15470[GenBank]). The 5-primer used (AGG ATG CAG TTC TTC GGA
CGC C) included the translation initiation codon ATG. The 3
-primer was
a reverse primer (CAG TTG ATG GAG CCA GTT GTC C) situated 223 bases
after the termination codon TGA. To determine the sequence of the
5
-end (primer region), we performed a standard rapid amplification of
cDNA ends-PCR (9). Briefly, cDNA was amplified by PCR using the
mouse reverse primer TTC CTA GGA GCT GTA GCA CCT G, which is equivalent
to nucleotides 598-619 in the hamster sequence. After attaching a
poly(A) tail to the 5
-end of the PCR product obtained, amplification
of this product was accomplished by PCR using a dT(18) primer and a
mouse reverse primer, ACT CCA GTT GGA AGA GCC GGA A, which is
equivalent to nucleotides 205-226 in the hamster sequence. A final
round of amplification was performed using the mouse reverse primer TTC CTC CCG GAC ACG TTC ACT T (equivalent to nucleotides 101-123 in the
hamster sequence). PCR conditions were as follows: denaturing, 95 °C
for 30 s; annealing, 55 °C for 45 s; extension, 72 °C
for 120 s; 40 cycles. A last extension step lasted 10 min. PCR
products were purified by agarose gel electrophoresis and sequenced by automatic DNA cycle sequencing (Applied Biosystems 373 automated DNA
sequencer, Perkin-Elmer).
The intracellular iPLA2 from P388D1 cells
was purified and characterized in our laboratory as an approximately
80-kDa protein (5). The iPLA2 purified by Tang et
al. (7) from CHO cells and the protein expressed by the cDNA
have a molecular size of 84.5 kDa. Besides this similarity, the
purification strategy used by Tang et al. (7) contains some
analogous steps to those used for the macrophage iPLA2 (5),
and the enzymatic profile of the CHO enzyme is comparable. Therefore,
we hypothesized that both proteins would share a high degree of
similarity if not identity. We began to address this hypothesis by
taking advantage of the polyclonal antibody generated by Tang et
al. (7) against the C-terminal portion of the CHO protein. The
antiserum recognized pure iPLA2 from P388D1
cells, as shown by immunoblot (Fig. 1, lane
2). Purified CHO iPLA2 was run in parallel as a
control and showed identical mobility, focusing both proteins in the
80-85-kDa molecular mass range (Fig. 1, lane 1).
To further establish a similarity between the two proteins, a series of
inhibition studies were conducted. We have previously shown that the
iPLA2 from P388D1 cells is potently and
irreversibly inhibited by bromoenol lactone (BEL). After a
preincubation time of 5 min at 40 °C, BEL inhibits pure
iPLA2 with an IC50 of about 60 nM
(10). Likewise, iPLA2 from CHO cells was inhibited in a
dose-dependent manner by BEL with an IC50 of
120 nM (Fig. 2A). This inhibition
was irreversible even after exhaustive dialysis of treated enzyme
compared with control (Fig. 2B). Also, P388D1 iPLA2 is reversibly inhibited by PACOCF3
(IC50 = 4 µM), and again CHO
iPLA2 was inhibited by the same reagent with an
IC50 of 3 µM (Fig. 3).
Since BEL is an irreversible inhibitor, it was possible to label the
macrophage iPLA2 by incubation with [3H]BEL
(10). However, labeling with BEL required the presence of active
enzyme, since the inhibitory agent is generated in situ from
BEL by the hydrolytic action of the enzyme on the lactone ring (10).
Indeed, in previous studies with the P388D1
iPLA2, DTNB prevented labeling of the enzyme with
[3H]BEL (10). Although DTNB also inhibits the CHO
iPLA2 (Fig. 4), we have employed herein the
irreversible inhibitor MAFP (11), because it is a much more specific
reagent than DTNB. MAFP (20 µM) completely inactivated
the CHO iPLA2 (Fig. 4) and prevented labeling of the enzyme
with [3H]BEL (Fig. 5). Moreover, in the
absence of MAFP treatment, autoradiographic analysis of
[3H]BEL-treated enzyme revealed a single spot at about 85 kDa. This is the same molecular size as that obtained for the
P388D1 iPLA2 utilizing a similar strategy (10)
as well as that found by immunoblot (Fig. 1).
The nucleotide sequence of the P388D1 iPLA2 was obtained by analyzing mRNA from these cells by PCR using primers based on the CHO iPLA2 sequence (Fig. 6). Comparison of the nucleotide sequence of the two proteins revealed a 92% sequence homology. Most of the changes of the nucleotide sequence occurred at the third nucleotide of the codon, reflecting the 95% identity at the amino acid level between two closely related species. These results further support the notion that the hamster iPLA2 cloned by Tang et al. (7) is the species equivalent to the murine enzyme purified and characterized by us.
Our previous studies on the P388D1 cell iPLA2 have suggested that this enzyme may play an important role in regulating fatty acid remodeling reactions in the cells (8, 12), and a similar role has subsequently been attributed to the iPLA2 activity present in rat pancreatic islets (13). This implies that the iPLA2 enzyme might ultimately regulate several key aspects of cell physiology, such as new membrane synthesis that allows cell proliferation, or fatty acid exchange within phospholipids that allows adaptive homeostatic changes. Although the discovery by Gross and co-workers (14) that some iPLA2s are potently and selectively inhibited by BEL has accelerated research on this enzyme, study of the iPLA2 is hampered by the fact that it is extremely difficult to obtain sufficient amounts of pure protein for biochemical and sequence analysis.
We have presented evidence herein that the enzyme purified and cloned by Tang et al. (7) is the equivalent in hamster to the enzyme we identified (15), purified (5), characterized (5, 10, 11), and studied at the cellular level (8, 12) in mouse P388D1 macrophages. By using a wide variety of approaches, we have found biochemical, immunological, pharmacological, and genetic similarities between the two proteins, strongly suggesting that the same molecular entity is expressed in different species. There is, however, an apparent difference between the two enzymes concerning ATP sensitivity. Tang et al. (7) failed to observe any ATP effect on a partially purified preparation of CHO iPLA2. We have confirmed this finding using the assay system employed for the P388D1 iPLA2. We have previously reported on the apparent activation of P388D1 iPLA2 by ATP (5). The effect was shown to depend on the presence of Triton X-100 in the assay system. This, along with the fact that several other nucleotides manifested the same effect (i.e. ADP, UTP, GTP) raised the possibility that it might not be relevant as a regulatory mechanism. We have new data with P388D1 iPLA2 showing that, rather than stimulating the iPLA2, the ATP stabilizes the enzyme and protects it from denaturation; hence, higher activity is found in the presence than in the absence of ATP.2 Considering the fact that the two proteins have slightly different amino acid sequences and purification schemes, it is possible that the CHO enzyme lacks the region with which ATP interacts. Alternatively, it is possible that the CHO enzyme is more resistant to denaturation than the P388D1 enzyme.
The availability of the iPLA2 cDNA and protein sequence of the 80-kDa iPLA2, which is now classified as a Group VI PLA2 (16), will allow new experimental avenues to be explored in defining the role(s) of iPLA2 in phospholipid metabolism and cellular function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U88624[GenBank].
We thank Richard Loo (University of California at San Diego) and Jin Tang (Genetics Institute) for providing pure iPLA2 from P388D1 macrophages and CHO cells, respectively.