(Received for publication, June 18, 1996, and in revised form, November 7, 1996)
From the Institutes for Environmental Medicine and
§ Human Gene Therapy, University of Pennsylvania Medical
Center, Philadelphia, Pennsylvania 19104, the ¶ Department of
Chemistry and Biochemistry, University of Delaware, Newark, Delaware
19716, and the
Kazusa DNA Research Institute, Kisarazu,
Chiba 292, Japan
A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.
Phospholipase A2 (PLA2)1 represents a diverse family of enzymes that hydrolyze the sn-2-fatty acyl or alkyl bond of phospholipids. The best characterized member of this family is a group of low molecular mass (~15 kDa) enzymes that require Ca2+ for catalytic activity and show maximal activity in an alkaline (pH 8.5) medium (1). This group of enzymes, called secreted PLA2 (sPLA2; types 1-3), include snake and bee venoms and mammalian pancreatic and synovial PLA2 enzymes. The more recently characterized 87-kDa cytoplasmic PLA2 (cPLA2) requires only µM Ca2+ for binding to the interface and shows maximal activity at neutral pH (2). This enzyme is widely distributed in cell types and may be specifically linked to eicosanoid metabolism. Finally, there is a group of enzymes (iPLA2) that do not require Ca2+ for maximal activity. As recently reviewed (3), few of these enzymes have been purified, and in contrast to sPLA2 and cPLA2, little molecular information is available. Therefore, although the iPLA2 enzymes are widely distributed, relatively little is known about their intragroup relationships and specific functions. Based on biochemical characteristics, Ackermann and Dennis (3) have divided the iPLA2 enzymes into three subgroups: lysosomal iPLA2, brush-border membrane iPLA2, and intracellular (cytosolic/membrane) iPLA2. A distinguishing characteristic of the lysosomal iPLA2 group is maximal activity in acidic medium, and accordingly, we have designated the enzyme in this report as aiPLA2.
iPLA2 activity in rat lung homogenates was first described ~25 years ago (4), and activity at acid pH was subsequently demonstrated in rabbit lung (5), rat granular pneumocytes (lung alveolar type II cells) (6), and rat and human alveolar macrophages (7, 8). Activity has been localized further to the lung lamellar bodies (the surfactant secretory organelle) and the lysosomal fraction (5, 7, 9). Under our assay conditions, lung aiPLA2 activity is inhibited by a phospholipid transition-state analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), while Ca2+-dependent PLA2 activity in the lung homogenate is insensitive (7). Using MJ33 as a probe, we previously purified a protein with aiPLA2 activity to apparent homogeneity (10). This protein had a molecular mass of ~15 kDa and a unique N-terminal amino acid sequence. Subsequent attempts to reproduce the isolation yielded similar enzyme activity, although the molecular mass of the isolated protein was ~26 kDa. Furthermore, the amino acid sequence of the larger enzyme, as described in this report, does not contain the previously determined N-terminal sequence. Consequently, we believe that aiPLA2 described in this report is different from the previously isolated protein.
Amino acid sequencing (35 residues) of tryptic digests of the 26-kDa protein isolated from rat lung revealed 100% identity to deduced amino acids from the nucleotide sequence (previously unpublished) of a clone isolated from a human myeloblast cDNA library (11). This report provides evidence that the protein expressed by this cDNA is aiPLA2 and provides the first molecular cloning for this group of enzymes.
Lipids were obtained from Avanti Polar Lipids (Birmingham, AL). All radiochemicals and x-ray film were purchased from DuPont NEN, and bisbodipy-C11-PC was from Molecular Probes, Inc. (Eugene, OR). Trifluoromethylarachidonoyl ketone (AACOCF3) was from Calbiochem; bromoenol lactone was from Cayman Chemical Co., Inc. (Ann Arbor, MI); and p-bromophenacyl bromide (pBPB), 2-mercaptoethanol, diethyl p-nitrophenyl phosphate (DENP), Naja naja PLA2, Sephacryl S-100, and phenyl-Sepharose CL-4B were from Sigma. DE52 was purchased from Whatman (Maidstone, United Kingdom). Molecular mass standards for SDS-PAGE and Transblot membrane were from Bio-Rad. Nitrocellulose membrane was from Schleicher & Schuell. Superscript reverse transcriptase and T4 DNA polymerase were from Life Technologies, Inc. Klenow enzyme was from Boehringer Mannheim. pBluescript SK+ vector was from Stratagene (La Jolla, CA). In vitro transcription and wheat germ in vitro translation kits were from Ambion Inc. (Austin, TX). Male Sprague-Dawley rats weighing ~200 g were obtained from Charles River Breeding Laboratories (Kingston, NY).
Isolation of Rat Lung aiPLA2Rats were
anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally).
Lungs were ventilated through the trachea, perfused through the
pulmonary artery in situ to clear them of blood, removed
from the thorax, and stored at 80 °C. Frozen lung tissue (~80 g)
was extensively homogenized with Polytron PT-10 and Potter-Elvehjem
homogenizers in 200 ml of ice-cold 50 mM Tris-HCl buffer
(pH 7.4) containing 1 mM EDTA, 0.2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, and 10% glycerol. All
subsequent steps of purification were performed at 4 °C. Assays for
PLA2 activity in the absence and presence of MJ33 utilized
the fluorescence assay with acidic (pH 4.0) Ca2+-free
buffer (see below).
The lung homogenate was spun at 10,000 × g to remove
cell debris and then centrifuged at 100,000 × g for
1 h. The supernatant was subjected to 55% ammonium sulfate
fractionation. The resultant precipitate was dissolved in 80 ml of
buffer and dialyzed extensively against 50 mM Tris-HCl (pH
7.4) containing 1 mM EDTA and 5% glycerol. This buffer was
used in all further steps of purification. The dialyzed sample was
centrifuged at 1500 × g for 15 min., and the supernatant was applied to a DE52 column (38 x 2.2 cm), which was
washed with 2 bed volumes of the same buffer at a flow rate of 60 ml/h
and then eluted with a linear gradient of 0-50 mM NaCl. The major aiPLA2 activity was recovered after the second
bed volume wash and was retarded with respect to the unbound protein
peak (Fig. 1). The fractions containing
aiPLA2 activity that was inhibited by MJ33 were pooled,
concentrated to 7 ml on an Amicon YM-10 membrane, applied to a
Sephacryl S-100 HR column (120 × 2.2 cm), and eluted with buffer
at a flow rate of 17 ml/h (Fig. 1). Fractions with MJ33-sensitive
aiPLA2 activity (3.5 ml each) were pooled and applied to a
second DE52 column (7 × 1.3 cm). The column was washed with 5 bed
volumes of buffer and then eluted with a linear gradient of 10-60
mM NaCl. aiPLA2 activity eluted between 20 and
40 mM NaCl (Fig. 1). The eluted protein fraction was
applied to a 2-ml phenyl-Sepharose column equilibrated with buffer
containing 1.5 M KCl. The column was eluted with a step
gradient of 1.5 to 0 KCl, followed by buffer and then water.
PLA2 activity was detected in the H2O wash,
which was analyzed by SDS-PAGE.
Amino Acid Sequencing of Rat Lung Protein
Protein samples were analyzed by SDS-PAGE (15% acrylamide) under reducing conditions using the Laemmli buffer system (12). Bands were first visualized with Coomassie Blue and then with silver staining. The protein was electroblotted onto polyvinylidene difluoride membrane (Transblot) in order to analyze for internal amino acid sequences. Selected protein bands were digested in situ with trypsin and then separated by high pressure liquid chromatography using a Zorbax column. Ten peaks were selected and subjected to mass spectral analysis. Based on this analysis, three peptides were selected and analyzed for their amino acid sequences by Edman degradation (13). These analyses were carried out in the Protein Microchemistry Laboratory of the Wistar Institute (Philadelphia, PA). The sequences were compared with the National Center for Biotechnology Information data base for similarity to known sequences.
Isolation and Sequencing of the cDNA CloneThe human
myeloid cell line KG-1 (CCL246 from the American Type Culture
Collection, Rockville, MD) was used as a source of mRNA. RNA
preparation, cloning, and sequencing of the HA0683 clone (referred to
as the KIAA0106 gene) have been described previously (11, 14). Briefly,
cytoplasmic RNA was prepared using a non-ionic detergent (15), followed
by chromatography on oligo(dT)-cellulose to isolate the
poly(A)+ RNA. First-strand synthesis was primed with a
poly(dT)-NotI primer oligonucleotide
(5-CTCTAGAGGCGGCCGC(T)34-3
) using Superscript reverse
transcriptase. Second-strand synthesis was performed as described (16),
repaired with T4 DNA polymerase, digested with NotI,
size-fractionated on a sucrose gradient, and ligated into a
NotI-EcoRV-cut pBluescript SK+
vector. Automated sequencing was performed by the dideoxy method (15)
using Chemical Robot DSP-240 (Seiko Instruments Inc., Tokyo) or
CATALYST800 (Applied Biosystems, Inc., Foster City, CA), and analysis
was performed using an Applied Biosystems 373A sequencer and the
Applied Biosystems sequence analysis system INHERIT. Large-scale preparations of plasmid were obtained by centrifugation on cesium chloride-ethidium bromide gradients (15).
The cDNA clone HA0683 was linearized by digestion with NotI, and capped mRNA was then synthesized by transcription using T7 RNA polymerase and 7mGpppG. The transcripts were purified by phenol/chloroform extraction and ethanol precipitation, quantitated by comparing intensity after ethidium bromide staining with control RNA with known concentration, checked for integrity by electrophoresis on a formaldehyde-agarose (1%) gel, and subsequently used as a template for translation in wheat germ or oocytes.
Wheat Germ in Vitro TranslationTranslation of the HA0683
mRNA transcript was performed in the wheat germ system according to
the manufacturer's protocol. In brief, RNA samples of up to 2 µg
were translated in a reaction mixture containing 50 mM
potassium acetate, amino acid master mixture (0.16 M
creatine phosphate, 0.5 mM methionine, 0.5 mM leucine, and a 1 mM concentration of the other amino
acids), and wheat germ extract. The total reaction volume was 50 µl.
Each experiment included a negative control in which no RNA was added and a positive control in which XeF-1 RNA, encoding Xenopus
elongation factor-1 (Mr 50,300) provided by
the supplier, was added. Reaction mixtures were incubated at 27 °C
for 60 min. The translated products were then assayed for
aiPLA2 activity.
Labeling of the HA0683 translation product in wheat germ extract was performed with the addition of 29.7 µCi of translation-grade [35S]methionine (1174 Ci/mmol) and a 1 mM concentration of the other amino acids. Otherwise, the reaction mixture was as described above. Small aliquots of translated proteins were heated at 95 °C for 2 min in the sample buffer containing 5% 2-mercaptoethanol and subjected to 15% SDS-PAGE together with prestained molecular mass standards. Following electrophoresis, the gels were destained, dried, and subjected to autoradiography.
Xenopus laevis Oocyte InjectionOocytes were injected with in vitro transcribed aiPLA2 mRNA (50-70 ng/oocyte) or an equal volume (50 nl) of deionized H2O (mock injection) and incubated generally for 48 h at 18 °C in modified Barth's solution (17), which contains 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, and 10 mM NaHEPES. Oocytes were washed with saline, homogenized in pH 4 buffer, and then assayed for PLA2 activity. Measurements were performed on groups containing at least five oocytes.
PLA2 Assay Using LiposomesEnzyme activity was measured at pH 4 (40 mM acetate buffer with 5 mM EDTA) using either a liposome-based radiochemical assay as described previously (9) or a fluorescence assay (bisbodipy-C11-PC) for rapid screening. For the radiochemical assay, the liposomal substrate was labeled with 1-palmitoyl-2-[9,10-3H]palmitoyl-sn-glycerol-3-phosphocholine ([3H]DPPC)/egg PC/egg phosphatidylglycerol/cholesterol in a molar ratio of 10:5:2:3. The specific activity of [3H]DPPC was 4400 dpm/nmol. Lipids were dried under N2, resuspended in isotonic saline, repeatedly freeze/thawed by alternating liquid N2 and warm H2O, and then extruded through a 100-µm membrane to generate unilamellar liposomes. The 250-µl PLA2 assay reaction volume contained 0.226 µmol of total lipid, generally in 50 mM sodium acetate buffer (pH 4) plus 1 mM EGTA. Incubation was at 37 °C generally for 1 h. Enzyme activity in the assay showed linear increase with incubation time between 15 and 120 min with both wheat germ- and oocyte-expressed proteins. The reaction was stopped by the addition of CHCl3/CH3OH (2:1, v/v); lipids were extracted by the method of Bligh and Dyer (18); and radiolabeled free fatty acids were separated by a two-step TLC procedure using hexane/ether/acetic acid as a solvent system (9). Authentic palmitic acid was co-chromatographed. The free fatty acid spots were identified using I2 vapor, scraped from the plate, and analyzed by scintillation counting using an internal standard for quench correction. Recovered dpm was corrected for blank values obtained in the absence of enzymes. PLA2 activity was calculated based on the specific radioactivity of DPPC. In some experiments, lipids were analyzed by TLC using CHCl3/CH3OH/NH4OH/H2O (65:35:2.5:2.5 by volume) (19) to separate lyso-PC in order to assay for PLA1 activity. In other experiments, liposomes were labeled with [choline-3H]DPPC in addition to the usual label, and the generation of labeled free fatty acid and lyso-PC was compared.
For the PLA2 fluorescence assay, the liposomal substrate was DPPC/bisbodipy-C11-PC/phosphatidylglycerol/cholesterol in a molar ratio 10:0.05:2:3, and total lipid was 0.171 µmol in 250 µl of sodium acetate (50 mM) plus EGTA (1 mM) buffer at pH 4. To stop the reaction, the medium was diluted to 2 ml with the assay buffer, and the fluorescent product was measured at 490 nm excitation and 520 nm emission. Standard curves at pH 4 and 8.5 were linear with bodipy-C11 fatty acid concentration up to 4 µM and were used to calculate PLA2 activity.
To test substrate specificity of PLA2, liposomes containing PC with various radiolabeled fatty acyl or alkyl constituents were prepared with lipids in a molar ratio of 10:2:3 for PC/phosphatidylglycerol/cholesterol. To test H+ dependence, PLA2 activity with the standard or a micellar assay (see below) was measured in buffers with varying pH plus or minus 10 mM CaCl2 (all with 1 mM EGTA). Varying pH buffers contained 50 mM glycine (pH 3), 50 mM acetate (pH 4-5), 50 mM MES (pH 6), or 50 mM Tris (pH 7-9). For evaluation of MJ33, the inhibitor was added (usually at 3 mol %) to the lipid mixture prior to generation of the liposomal substrate. The effects of potential PLA2 inhibitors (AACOCF3, BEL, and DENP) were determined following preincubation of enzyme with inhibitor at pH 4 for 30 min at 37 °C. To determine the effect of pBPB, protein was preincubated with inhibitor at pH 7.4 in 25 mM Tris containing 0.5 mM EDTA. The concentrations selected for testing of inhibitors have been shown previously to produce maximal effect in other systems (9, 20, 21).
PLA2 Assay Using Mixed MicellesMixed micelles were prepared with 10 mM [3H]DPPC (specific activity, 4400 dpm/nmol) and 4 mM Triton X-100 in saline by sonication using a cuphorn sonicator (Heat Systems Ultrasonics, Plainview, NY) at 70% of maximum power with 3 × 20-s bursts at 30-s intervals. For PLA2 assay, 50 µl of enzyme, 850 µl of buffer, and 100 µl of micelles (final DPPC concentration, 1 mM) were incubated at 37 °C for 60 min in the presence or absence of Ca2+. Lipid radioactivity was assayed as described above for the liposomal assay.
Lysophospholipase AssayTo measure lysophospholipase activity, the [3H]lyso-PC substrate was generated by treating liposomes labeled with [choline-3H]DPPC with N. naja PLA2 at pH 8.5 plus 10 mM Ca2+ to achieve complete hydrolysis. [3H]Lyso-PC was separated from the incubation mixture by TLC, and the lyso-PC spot was scraped and extracted with CHCl3/CH3OH (20:1) (19). Enzyme was incubated with labeled lyso-PC as substrate at pH 4 or 8.5 for 1 h at 37 °C. Lipids were extracted by the method of Bligh and Dyer (18), and the aqueous fraction was assayed for radioactivity (representing [3H]glycerophosphorylcholine or its degradation products).
PAF Acetylhydrolase ActivityThe incubation medium contained 50 mM Tris-HCl (pH 4 or 7.4), 5 mM EDTA, and 20 nmol of [acetyl-3H]PAF in a total volume of 250 µl. After 60 min of incubation at 37 °C, the reaction was stopped by the addition of 2.5 ml of CHCl3/CH3OH (4:1) and 250 µl of H2O (22). The aqueous phase was assayed for radioactivity to determine the amount of acetate liberated.
Northern Blot AnalysisGranular pneumocytes were prepared
by elastase digestion of rat lungs as described previously (23). Total
RNA was extracted from freshly isolated granular pneumocytes or from
homogenized rat lungs using the acid guanidinium
thiocyanate/phenol/chloroform extraction method (24). The RNA was
dissolved in diethyl pyrocarbonate-treated H2O, quantitated
by absorbance at 260 nm, and stored frozen at 80 °C. Extracted RNA
samples were electrophoresed on 1% agarose gels containing
formaldehyde (15). The size-separated RNA was capillary-transferred
onto nitrocellulose membranes using 20 × SSC (1 × SSC = 150 mM NaCl and 15 mM sodium citrate). After
an overnight transfer, the RNA was fixed by baking for 2 h at
80 °C in a vacuum oven. [32P]dCTP (3000 Ci/mmol) was
used to generate 32P-labeled HA0683 cDNA probes by
random priming using Klenow enzyme. Membranes were prehybridized in a
solution consisting of 5 × SSC, 5 × Denhardt's solution
(15), 50% formamide, 1% SDS, and 10 µg/ml denatured salmon sperm
DNA at 42 °C for at least 4 h. Hybridization with the probe
(5 × 105 cpm/ml) was performed by overnight
incubation in the same buffer at 42 °C. Membranes post-hybridization
were washed once with 2 × SSC and 0.1% SDS for 15 min at room
temperature and twice with 0.1 × SSC and 0.1% SDS at 65 °C
for 30 min and then exposed to x-ray film.
By the assay used to identify fractions during the isolation procedure, the isolated rat lung protein was active at acid pH in the absence of Ca2+ and was inhibited by MJ33. The sequential use of the DE52, Sephacryl S-100, and repeat DE52 columns resulted in a 163-fold increase in PLA2 specific activity, although 99% of the starting activity was lost (Table I). The subsequent phenyl-Sepharose column resulted in further purification as determined by SDS-PAGE (Fig. 2), although insufficient protein was recovered for enzymatic characterization. Enzymatic properties of the preparation were analyzed with the partially purified protein from the second DE52 column.
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Identification of the Human cDNA Clone Corresponding to Rat Lung aiPLA2
SDS-PAGE of the fraction with
aiPLA2 activity that eluted from the phenyl-Sepharose
column showed two proteins bands with apparent molecular masses of 26.3 and 25.0 kDa (Fig. 2), and these were used for internal amino acid
sequencing. Fragments produced by tryptic digests of these two proteins
were subjected to amino acid sequence analysis. For the 25.0-kDa
protein, two fragments (25 amino acids total) were sequenced and found
to have significant homology to rat brain thiol-specific antioxidant
(25). Therefore, this protein was not further evaluated. For the
26.3-kDa protein, three internal peptides comprising 35 amino acids
were sequenced and showed 100% identity (Fig. 3) to a reported
translated open reading frame of unknown function from a human male
myeloblast cell line, KG-1 (11). Fig. 3 illustrates the
nucleotide sequence of this 1653-base pair cDNA (HA0683) and its
deduced amino acid sequence comprising 224 residues (calculated
Mr = 25,032). In addition to the putative
protein coding sequence, this clone contains 44 base pairs of upstream
and 934 base pairs of downstream sequence (Fig. 3).
Expression in Wheat Germ in Vitro Translation System
To
determine if the human cDNA encodes a translatable protein with a
molecular mass similar to that of rat lung protein, HA0683 cDNA was
transcribed in vitro with T7 polymerase and translated in vitro using a wheat germ system. SDS-PAGE with
autoradiography of the translated [35S]methionine-labeled
protein (Fig. 4) showed an apparent molecular mass of
25.8 kDa, similar to the mass of the predicted protein and of the
PLA2 enzyme isolated from rat lung (Fig. 2).
[35S]Methionine-labeled protein expressed by the wheat
germ system increased as a function of RNA concentration, with
saturation at ~1.0 µg in a 50-µl reaction volume (Fig. 4).
Saturation of the wheat germ system at higher concentrations of RNA has
been shown previously (26).
Ten separate in vitro translations were carried out with the
wheat germ expression system; although these used varying
concentrations of cRNA, in each we found the expression of an enzyme
activity with the characteristics of aiPLA2 (assay at pH 4 in Ca2+-free buffer). PLA2 activity of the
expressed protein measured by the radiochemical assay increased
linearly as a function of the cRNA concentration used for expression up
to 1 µg (Fig. 5). For translation with 1 µg of cRNA,
expressed PLA2 activity was 1556 ± 20 pmol/h
(mean ± S.E.; n = 7). Control incubation with the
wheat germ system in the absence of cRNA showed zero PLA2 activity. To confirm PLA2 activity, generation of free
fatty acid from [3H]palmitoyl-labeled DPPC and of
[3H]lyso-PC from [3H]choline-labeled DPPC
was measured in the same assay using protein expressed in the wheat
germ system. Liberation of the free fatty acid was 1288 ± 32 pmol/h, and generation of lyso-PC was 1273 ± 27 pmol/h (mean ± range; n = 2). Blank values for PLA2
assays were ~250 dpm, and the usual value for expressed enzyme was
~6000 dpm, giving a satisfactory signal-to-noise ratio of ~24.
Characterization of aiPLA2 Activity
The standard
PLA2 assay was carried out in the absence of
Ca2+. The activities of both the expressed PLA2
at pH 3-8.5 as well as the partially purified rat lung enzyme at pH 4 were not affected by the addition of Ca2+ (Fig.
6). The pH versus activity profile for the
isolated lung enzyme showed maximal activity at pH 4 and essentially no
activity at pH 6 and above (Fig. 6). The pH profile for the expressed
protein was similar (Fig. 6). The pH profile and Ca2+
independence for the expressed protein were similar using either the
liposomal or micellar assay system (Fig. 6).
aiPLA2 expressed in vitro showed similar
activity using dipalmitoyl-PC or palmitoylarachidonoyl-PC as substrate
(Fig. 7 and Table II). The apparent
Km for the substrates was ~250 µM.
PLA2 activity using the alkyl ether phospholipid,
O-hexadecylarachidonoyl-PC, as substrate at 1 mM
was lower by ~50% compared with DPPC (Table II), although the
apparent Km was similar (Fig. 7). In vitro translated aiPLA2 did not show any
PLA1 or lysophospholipase activity, and PAF acetylhydrolase
activity was negligible (Table II).
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Potential inhibitors (AACOCF3 (100 µM), BEL (100 µM), pBPB (20 µM), and 2-mercaptoethanol (5 mM)) were tested for their effects on the activity of the isolated rat lung enzyme and in vitro translated aiPLA2 (Table III). ATP (10 mM), an activator of myocardial iPLA2 (27), was also evaluated. None of these agents had a significant effect on the activity of the rat lung or expressed enzyme. MJ33 significantly inhibited the activity of aiPLA2 (Table III), with a maximal effect at 1 mol % (Fig. 8). Because of the "lipase" motif (Fig. 3), the effect of the serine protease inhibitor DENP was evaluated and was found to significantly inhibit aiPLA2 activity, with 80% inhibition at 0.5 mM (Fig. 8 and Table III).
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Expression of Human aiPLA2 in Xenopus Oocytes
To
further evaluate the protein product of the HA0683 clone, cRNA
generated from the clone was expressed in Xenopus oocytes and assayed for aiPLA2 activity. Expressed
aiPLA2 activity increased as a function of time after
oocyte injection, reached a maximum at 30 h, and maintained this
level at 48 h post-injection (Fig. 5). Further studies were
carried out using oocytes at 48 h after injection. In each of
seven separate injection experiments, we observed an increase in
aiPLA2 activity compared with oocytes injected with
deionized H2O, with a mean increase of ~40% (Table IV). Similar results were obtained using both the
fluorescence and radiochemical assays. For the radiochemical assay, the
blank value (~250 dpm representing 50 dpm/oocyte) was subtracted
from the measured PLA2 activity; this gave a
signal-to-noise ratio of ~10 for the expressed PLA2
activity or ~4 for the difference between cRNA- and deionized
H2O-injected oocytes. To confirm PLA2 activity,
liberation of free fatty acid and generation of lyso-PC were compared
in the same assay; liberation of free fatty acid was 590 ± 13 dpm/oocyte, and generation of lyso-PC was 580 ± 6 dpm/oocyte
(mean ± range; n = 2). Activity was not changed
in the presence of 10 mM Ca2+, but was
decreased to the basal value in the presence of 3 mol % MJ33 (Table
V). When assayed at pH 8.5 in the presence of
Ca2+, activity decreased by 90% compared with pH 4. These
results parallel those obtained with the wheat germ expression system and the isolated lung enzyme.
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Since the cDNA clone was isolated from
a human myeloblast cell line, it was of interest to test whether this
mRNA is present in rat lung, the source of the original protein
isolation. Northern analysis of aiPLA2 RNA demonstrated
high levels of expression of a hybridizing mRNA, ~1.7 kilobase
pairs in size, in both rat lung and granular pneumocytes isolated from
rat lung (Fig. 9).
iPLA2 enzymes have been shown to be widely distributed and ubiquitously expressed in most mammalian tissue, underlining their potential importance in cellular functions (3). Although mammalian iPLA2 enzymes have been isolated and characterized from various sources, there is scant molecular information due to difficulties associated with purification and insufficient yield. To date, the only iPLA2-type enzyme that has been sequenced is a PAF hydrolase (28), which also has properties of a low density lipoprotein-associated PLA2 (29). Therefore, little is known about the structure or mechanisms of iPLA2 enzymes or their relationship to other PLA2 enzymes. Here, we describe the cloning, sequencing, and characterization of a human iPLA2 (aiPLA2) that shows maximal activity in an acidic medium.
Evidence for aiPLA2 Activity of Expressed ProteinExpression of the HA0683 cDNA using both the wheat germ and Xenopus oocyte systems demonstrated PLA2 activity. In vitro translated aiPLA2 did not show any PLA1 or lysophospholipase activity, excluding the possibility that the combined activities of those two enzymes could have accounted for the measured PLA2 activity. Furthermore, assays of activity with both wheat germ- and oocyte-expressed enzyme based on recoveries of labeled free fatty acid from sn-2-fatty acyl-labeled DPPC and labeled lyso-PC from choline-labeled DPPC were nearly identical, indicating PLA2 for the lipolytic activity. The pH 4 optimum for activity and the Ca2+ independence indicated that the protein encoded by the HA0683 cDNA clone has enzymatic properties that correspond to the activity profile for the partially purified rat lung enzyme as well as the activity previously demonstrated in homogenates of rat lungs and granular pneumocytes (7, 30). Northern analysis demonstrated that this gene is highly expressed in rat lung and granular pneumocytes (Fig. 9). aiPLA2 activity was significantly inhibited by MJ33, a competitive inhibitor of acidic Ca2+-independent PLA2 activity in lung homogenates and subcellular organelles (lysosomes and lamellar bodies) (7, 30), and by a serine protease inhibitor, DENP. Unlike other iPLA2 enzyme activities that have been characterized, the activity of the expressed protein was not affected by the presence of detergent (Triton X-100), and neither it nor the isolated rat lung enzyme was affected by the inhibitor BEL or the activator ATP (27, 31). Thus, it appears that aiPLA2 presented in this report is distinct from the iPLA2 enzymes previously purified from the P388D1 macrophage cell line (20) and from cardiac muscle (27). The relative activity of PAF acetylhydrolase, a Ca2+-independent enzyme that can cleave short-chain acyl groups at the sn-2-position (22), was negligible. The isolated rat enzyme and the expressed protein are differentiated from sPLA2 and cPLA2 by their Ca2+ independence and pH profile as well as by insensitivity to inhibitors (pBPB, 2-mercaptoethanol, and AACOCF3).
Properties of the Predicted ProteinThe HA0683 cDNA encodes a mature protein of 224 amino acids with a calculated molecular mass of 25.0 kDa. We have shown in a wheat germ in vitro translation system that mRNA transcribed from this clone results in the expression of a protein of 25.8 kDa in size, in reasonable agreement with the predicted mass of the deduced amino acid sequence and with the estimated mass of aiPLA2 isolated from rat lung. The predicted protein has 32 (14.3%) negatively charged and 30 (13.4%) positively charged residues, with no predicted charge clusters and a predicted pI of 6.0 (32). Nonpolar residues account for 107 (48%) of the 224 amino acids in the predicted sequence. A hydrophobicity plot of the predicted protein did not indicate any extended regions of high hydrophobicity, consistent with the fact that aiPLA2 was isolated as a soluble protein.
Sequence Homology to Other ProteinsSearches for similarity to the HA0683 cDNA sequence at the protein (SWISSPROT protein data base) and DNA (GenBankTM data base) levels did not identify any phospholipases. Therefore, aiPLA2 is apparently not related to any of the phospholipases previously sequenced. The predicted aiPLA2 enzyme contains a 5-amino acid sequence, GXSXG (double underline in Fig. 3), that has been described as a lipase motif and may represent the active serine of the catalytic triad SDH (33). Inhibition by DENP supports a serine-based mechanism for aiPLA2 activity. The motif has been described in serine proteases and in neutral lipases. Of note, it has also been described in PAF acetylhydrolase (low density lipoprotein-associated PLA2) (28, 29). Thus, the motif is present in both iPLA2 enzymes (aiPLA2 and PAF acetylhydrolase) that have been cloned, although it is not observed in sPLA2 or cPLA2.
From the protein data base, the amino acid sequence with the most similarity to an HA0683-encoded protein was a hypothetical 29.5-kDa protein from yeast (SWISSPROT ID YBG4) (34), which matched at 99 out of 261 amino acid positions. A search for similarity to the deduced N-terminal sequence of aiPLA2 revealed homology to two human unknown proteins isolated by two-dimensional gel electrophoresis from liver (SWISSPROT ID P30041) (35) and red blood cells (SWISSPROT ID P32077) (36). These sequences, consisting of 14 and 12 amino acids, respectively, showed 100% homology to the predicted aiPLA2 sequence following its deduced start site. Electrophoresis showed molecular masses of 23.5 kDa for the liver protein (35) and 26 kDa for the red blood cell protein (36), with an identical pI of 6.2. The similarities in N-terminal sequence, size, and pI to the parameters predicted for the HA0683-encoded protein suggest that these human proteins of unknown function may represent aiPLA2, indicating widespread distribution for the enzyme. Although originally isolated from a human myeloid cell line, HA0683 hybridized to RNA from all tissues and cell lines tested (11). Since aiPLA2 may be a lysosomal enzyme, its widespread distribution would be expected.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D14662[GenBank].
We thank Dr. D. Speicher for assistance with amino acid analysis and interpretation.
We have now cloned and sequenced the
actual 5 end of the aiPLA2 RNA using a 5
rapid
amplification of cDNA ends (Frohman, M. A. (1993) Methods
Enzymol. 218, 340-358) and found an additional 24 nucleotides but no upstream ATG translational start codon.