(Received for publication, June 19, 1995; and in revised form, July 20, 1995)
From the
Platelet-activating factor (PAF) acetylhydrolase, which
inactivates PAF by removing the acetyl group at the sn-2
position, is distributed widely in plasma and tissues. In a previous
study, we demonstrated that the PAF acetylhydrolase activity present in
the soluble fraction of bovine brain cortex could be separated
chromatographically into three peaks (tentatively designated isoforms
Ia, Ib, and II) (Hattori, M., Arai, H., and Inoue, K.(1993) J.
Biol. Chem. 268, 18748-18753). In this study, these three
isoforms were also detected in kidney and liver cytosols, although
their relative activity ratios in these tissues differed. In
particular, isoform II was responsible for the majority of the bovine
liver PAF acetylhydrolase activity. We purified isoform II from bovine
liver cytosol to near homogeneity and demonstrated that it is a single
40-kDa polypeptide. This enzyme was inactivated by diisopropyl
fluorophosphate and 5,5`-dithiobis(2-nitrobenzoic acid), suggesting
that both serine and cysteine residues are required for the enzyme
activity, and [H]diisopropyl fluorophosphate
labeled only the 40-kDa polypeptide, confirming the enzyme's
identity. Isoform II showed a comparatively broader substrate
specificity than isoform Ib. Isoform II hydrolyzed propionyl and
butyroyl moieties at the sn-2 position approximately half as
effectively as it did PAF, whereas isoform Ib hardly hydrolyzed these
substrates.
Taken together with previous data, the current findings indicate that tissue cytosol contains at least two types of PAF acetylhydrolase with respect to polypeptide composition, substrate specificity, and tissue distribution and suggest that these two enzymes may share distinct physiological functions in tissues.
Platelet-activating factor
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) ()is a potent inflammatory mediator that causes
microvascular leakage, vasodilation, smooth muscle contraction,
endothelial adhesion, and neutrophil, macrophage, and eosinophil
activation (for review, see (1, 2, 3, 4, 5) ). PAF is
produced by a variety of cells and
tissues(6, 7, 8) , and its synthesis by a
variety of mammalian cells has been examined and is known to be tightly
regulated(9) . Its inactivation is catalyzed by a specific
acetylhydrolase, which removes the acetyl group at the sn-2
position of the glycerol backbone, to produce the biologically inactive
lyso-PAF(10, 11) . This process can also regulate PAF
accumulation in certain types of cell. For example, human macrophages
regulate PAF accumulation by altering intracellular PAF acetylhydrolase
activity levels(12) . It was also demonstrated that 10-12
times more PAF was produced when platelets were pretreated with
phenylmethanesulfonyl fluoride, an inhibitor of intracellular PAF
acetylhydrolase, and stimulated with thrombin than after thrombin
stimulation alone(13) .
PAF acetylhydrolase activity was
examined in various animal tissues and shown to be present in most of
rat tissues(11, 14, 15, 16) . It has
been proposed that there is a family of distinct enzymes in animal
tissues and cells(14, 17) . Recently, PAF
acetylhydrolase was purified from human erythrocytes(18) . This
enzyme has a molecular mass of 25 kDa and behaves as a homodimer. We
found that at least three isoforms (tentatively designated Ia, Ib, and
II) of PAF acetylhydrolase existed in bovine brain and that isoform Ib,
which was the most abundant, was a heterotrimeric enzyme composed of
29-, 30- and 45-kDa subunits(19) . Of these subunits, the
29-kDa one served as the catalytic center, as only this subunit was
labeled by [H]diisopropyl fluorophosphate (DFP),
a potent inhibitor of the enzyme(19, 20) . The amino
acid sequence deduced from its cDNA is unique and not homologous with
those of any other known phospholipases and lipases.
In the current study, we purified another isoform (isoform II) of PAF acetylhydrolase from bovine liver and showed that intracellular PAF acetylhydrolases Ib and II are distinct proteins with different substrate specificities and tissue distributions.
Next, these soluble fractions were subjected to DEAE-Sepharose followed by hydroxylapatite column chromatography (Fig. 1). The kidney and liver PAF acetylhydrolase activities separated into three peaks, as did brain PAF acetylhydrolase activity. Activity was present in two peaks among the fractions eluted by about 150 and 300 mM NaCl from the DEAE column (corresponding to peaks I and II, respectively, of brain PAF acetylhydrolase), although peak I of the liver fraction was very small. The peak I fractions of liver and kidney were both separated into two peaks by hydroxylapatite chromatography (corresponding to peaks Ia and Ib of brain PAF acetylhydrolase). Therefore, these data indicate that bovine kidney and liver possess the same set of isoforms as bovine brain. In contrast to the brain, abundant peak II activity was present in the soluble fractions of kidney and liver. Remarkably, peak II was responsible for most of the PAF acetylhydrolase activity in the liver. No other peak showing activity was observed in the soluble fractions of the kidney or liver during the sequential chromatography.
Figure 1:
Distribution of three PAF
acetylhydrolase isoforms in bovine brain, kidney, and liver. Soluble
fractions (100 ml) of bovine brain (A), kidney (C),
and liver (E) were applied to a DEAE-Sepharose CL-6B column
(2.5 cm 20 cm) equilibrated with 10 mM Tris-HCl (pH
7.4), 1 mM EDTA, and 10% (v/v) glycerol, and eluted with a
350-ml linear NaCl gradient (0-400 mM). Peak I fractions
of brain (B), kidney (D), and liver (F) from
the DEAE-Sepharose column were pooled and applied to a hydroxylapatite
column (1.5 cm
20 cm) equilibrated with 10 mM
KH
PO
-KOH (pH 6.8), 5 mM 2-ME, and 10%
(v/v) glycerol, and eluted with a 160-ml linear KH
PO
gradient (10-200 mM). The hydrolytic activity with
PAF as the substrate was measured as described under
``Experimental Procedures'' using a 20-µl aliquot of each
fraction.
Figure 2: Elution profile of PAF acetylhydrolase II on a Mono Q FPLC column. The active fractions from the hydroxylapatite column were pooled (about 25 ml), dialyzed against 500 ml of buffer C, and then applied to a Mono Q HR 5/5 FPLC column equilibrated with buffer C. After washing the column with buffer C, elution was carried out with an 18-ml linear gradient of NaCl (0-500 mM) in buffer C. Fractions (0.3 ml) were collected, examined for PAF acetylhydrolase activity, and subjected to SDS-PAGE, after which the gel was stained with Coomassie Brilliant Blue
Sodium fluoride has been reported to inhibit partially rat lung cytosolic PAF acetyl hydrolase(23) , and Stafforini et al. (17) have shown that PAF acetylhydrolase in human erythrocytes is very sensitive to sodium fluoride. We tested the effect of sodium fluoride on purified PAF acetylhydrolase II, which we found was quite resistant to this agent over a wide range of concentrations (data not shown), in agreement with the observation of Stafforini et al. (17) that PAF acetylhydrolase activity in rat liver is resistant to sodium fluoride.
Figure 3:
[H]DFP labeling of
PAF acetylhydrolase II. PAF acetylhydrolase II was partially purified
from various tissues by sequential column chromatography using
butyl-Toyopearl, DEAE- Sepharose, and Bio-Gel A-0.5m columns. Then, 5
nmol/min enzyme were incubated with [
H]DFP (5
µCi, 0.58 nmol) at 25 °C for 30 min and subjected to SDS-PAGE. A, Coomassie Brilliant Blue-stained gel after SDS-PAGE of the
purified PAF acetylhydrolase II from liver. B, fluorograph of
labeled PAF acetylhydrolase II from liver. C, fluorograph of
labeled PAF acetylhydrolase II from brain. D, fluorograph of
the labeled PAF acetylhydrolase II from kidney. For details, see
``Experimental Procedures.''
Figure 4: Reverse-phase HPLC elution profile of peptide fragments derived from purified PAF acetylhydrolase II. Purified PAF acetylhydrolase II was digested with lysyl endopeptidase and the reaction mixture was subjected to reverse-phase HPLC. For details, see ``Experimental Procedures.''
Figure 5:
Different substrate specificities of PAF
acetylhydrolases Ib and II. The hydrolytic activities using various PAF
derivatives as substrates were measured as described under
``Experimental Procedures.'' Purified PAF acetylhydrolases Ib
and II (0.3 nmol/min) were incubated with 80 µM
radiolabeled substrate dissolved in 250 µl of 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 5 mM 2-ME. The
reaction was stopped by adding 2 ml of
CHCl/CH
OH (1:1 v/v), the lipids were extracted
as described by Bligh and Dyer(22) , spotted onto a silica gel
plate, and developed with
CHCl
/CH
OH/CH
COOH/H
O
(25:15:4:2, v/v). Each spot of reaction product or undegraded substrate
was scraped off and transferred to a scintillation vial, and the
radioactivity was measured. A, PAF; B, propionyl-PAF; C, butyroyl-PAF; D, succinoyl-PAF; E,
glutaroyl-PAF.
In the current study, we purified another type of
intracellular PAF acetylhydrolase from bovine liver and showed that it
was a single 40-kDa polypeptide. Moreover, we found that the relative
ratios of these two enzymes in bovine tissues differed. We demonstrated
previously that the PAF acetylhydrolase activity present in the
cytosolic fraction of bovine brain cortex separated chromatographically
into three peaks (designated peaks Ia, Ib, and II) and that the enzyme
in peak Ib was a heterotrimer comprising 29-, 30- and 45-kDa
polypeptides(19) . The enzyme belong to a novel serine esterase
family: both the 29- and 30-kDa subunits have a catalytic serine
residue (20) . ()In contrast, PAF acetylhydrolase II
(in the peak II fraction) appears to be a monomeric enzyme. It is,
however, too early to be absolutely certain that this is the case, as
we could obtain only a sharp single peak of activity by gel filtration
chromatography in the presence of a certain detergent (CHAPS).
Consistent with these data, Stafforini et al. (14) showed that PAF acetylhydrolase from rat liver cytosol was
eluted in a relatively broad peak when subjected to Sephadex G-200 gel
filtration chromatography. The chromatographic behavior on a
butyl-Toyopearl hydrophobic column suggested that PAF acetylhydrolase
II is more hydrophobic in nature than PAF acetylhydrolase Ib. The
former enzyme may be associated with some other cytosolic component(s).
It should be noted that human plasma PAF acetylhydrolase, a 44-kDa
monomer, is associated with plasma lipoproteins, such as low and high
density lipoproteins, and cannot be dissociated from them without
detergent (24, 25) .
The suggestion that PAF acetylhydrolases Ib and II are distinct enzymes is also supported by the amino acid sequence data. The catalytic components of PAF acetylhydrolase Ib, the 29- and 30-kDa subunits, are novel proteins with no significant homology with other known proteins. The partial amino acid sequence of the 40-kDa PAF acetylhydrolase II matches neither those of these polypeptides nor any other protein reported so far. Very recently, the cDNA for human plasma PAF acetylhydrolase was cloned and sequenced(26) . Both plasma and intracellular (isoform Ib) enzymes belong to the serine esterase family, but otherwise these proteins showed no sequence similarities, and no identical sequences in the plasma enzyme and intracellular enzyme isoform II were observed. Northern blot analysis of human tissues using the cDNA for the plasma enzyme revealed that signals was hardly detected in the human liver or kidney, whereas in both these bovine tissues, PAF acetylhydrolase II is the most abundant isoform. These findings also support the hypothesis that the plasma enzyme and isoform II are distinct proteins. The N-terminal amino acid sequence of PAF acetylhydrolase from human erythrocytes has been reported(18) . Despite repeated attempts to determine the N-terminal amino acid sequence of the purified PAF acetylhydrolase II, we were unable to do so, possibly because some modification of the N-terminal amino group occurred. The erythrocyte enzyme appears to be a homodimer consisting of the 25-kDa polypeptide(18) . Moreover, the elution positions of the liver and erythrocyte PAF acetylhydrolases indicated that these two activities could be differentiated from each other. All these data support the concept that PAF acetylhydrolase from erythrocyte cytosol is not identical to PAF acetylhydrolase II. Aarsman et al. (27) reported that two lysophospholipases (I and II) existed in bovine liver cytosol and that lysophospholipase II, the molecular mass of which SDS-PAGE showed was 63 kDa, had intrinsic PAF acetylhydrolase activity, although deacetylation of PAF was five times slower than deacylation of lysophospholipid. We tested whether the purified PAF acetylhydrolase II possessed lysophospholipase activity using 1-palmitoyl-glycerophosphocholine as a substrate and found that this enzyme was unable to split acyl chains attached to the sn-1 position of glycerophospholipid. According to our purification data, over 70% of the original activity was lost during the first chromatographic step, when lysophospholipase II might have been separated from PAF acetylhydrolase II.
Recently, we found that peak
Ia obtained after hydroxylapatite chromatography of peak I was a 60-kDa
enzyme consisting of 29- and 30-kDa polypeptides. These polypeptides
are immunochemically identical to the corresponding subunits of isoform
Ib, indicating that these two isoforms are mutually
related. Thus, at present, it can be concluded that the intracellular
type of PAF acetylhydrolase comprises at least three groups of enzymes,
PAF acetylhydrolases I (Ia and Ib), PAF acetylhydrolase II, and
erythrocyte-type PAF acetylhydrolase.
We showed previously that one subunit (45 kDa) of isoform Ib is identical to the product of the causative gene for Miller-Dieker lissencephaly, a human brain malformation that manifests a smooth cerebral surface and abnormal neuronal migration(28) . This study raised the possibility that the migration of neuronal cells during brain development is regulated by PAF (or a PAF-like phospholipid), and a defect in PAF acetylhydrolase Ib results in defective or unregulated PAF metabolism in the cells. It should be noted that the catalytic subunit of isoform Ib possesses a sequence of about 30 amino acids located 6 residues downstream from the active serine site, which exhibits significant homology to the first transmembrane region of the PAF receptor(20) , whereas plasma PAF acetylhydrolase does not possess such a region(26) . Although it is not clear whether this domain is involved in substrate binding, such a sequence in the catalytic subunit may confer the property of specific recognition of PAF on isoform Ib. The function of isoform II is unknown at present. Isoform II was found to have a relatively broad substrate specificity compared with isoform Ib; the former hydrolyzed butyl, propyl, succinoyl, and glutaroyl groups attached to the sn-2 position of PAF appreciably, but the latter did not. The substrate specificity of isoform II resembles that of plasma PAF acetylhydrolase rather than isoform Ib. A potential role of PAF acetylhydrolase is thought to be degradation of toxic products of lipid peroxidation, such as oxidatively fragmented phospholipids(18, 19, 29, 30, 31) . Since erythrocytes have no ability to produce PAF, it is reasonable to infer that the function of erythrocyte PAF acetylhydrolase may be to scavenge oxidatively damaged phospholipids produced in the cells. Intracellular PAF acetylhydrolase II may also provide a mechanism to protect the tissues against oxidative damage, as it would recognize and degrade oxidized species without acting on structural membrane phospholipids. In this context, it would be interesting to consider that the liver and kidney, both of which are abundant in isoform II, are known to be rich in superoxide dismutase. The next challenge is to clarify the structural and functional differences of the two distinct intracellular PAF acetylhydrolases.