Paraoxonase-1 (PON1), an high density
lipoprotein (HDL)-associated organophosphate triesterase,
suppresses atherosclerosis in an unknown way. Purified PON1 protects
lipoprotein particles from oxidative modification and hydrolyzes
pro-atherogenic oxidized phospholipids and the inflammatory mediator
platelet-activating factor (PAF). We find human PON1 acted as a
phospholipase A2 but not as a phospholipase C or D
through cleavage of phosphodiester bonds as expected. PON1 requires
divalent cations, but EDTA did not block the phospholipase
A2 activity of PON1. In contrast, a serine esterase
inhibitor abolished phospholipase activity even though PON1 has no
active-site serine residues. PAF acetylhydrolase, an oxidized
phospholipid phospholipase A2, is a serine esterase associated with specific HDL particles. Western blotting did not reveal
detectable amounts of PAF acetylhydrolase in PON1 preparations, although very low amounts of PAF acetylhydrolase might still account for PON1 phospholipase A2 activity. We revised the standard
PON1 purification by first depleting HDL of PAF acetylhydrolase to find
PON1 purified in this way no longer hydrolyzed oxidized phospholipids or PAF. Serum from a donor with an inactivating mutation in the PAF
acetylhydrolase gene did not hydrolyze oxidized phospholipids or PAF,
yet displayed full paraoxonase activity. We conclude that PAF
acetylhydrolase is the sole phospholipase A2 of HDL and
that PON1 has no phospholipase activity toward PAF or pro-atherogenic oxidized phospholipids.
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INTRODUCTION |
Paraoxonase-1 (PON1)1
catalyzes the hydrolysis of organophosphorous triesters and aryl
carboxyl esters; it is of interest because of its hydrolysis of
paraoxon (a metabolite of the insecticide parathion) and neurotoxins
such as sarin and diisopropylfluorophosphate. Insects and birds, in
contrast to mammals, lack this activity, rendering them sensitive to
various organophosphates (1). Hydrolysis of these substrates by PON1 is
Ca2+-dependent, and the enzyme is
extraordinarily sensitive to EDTA (2); for instance EDTA used as an
anti-coagulant destabilizes and irreversibly inactivates the enzyme
(3). PON1 is of interest for a second reason documented in a broad
range of literature (4-13) that associates PON1 with a suppression
of atherogenesis. For example, mice lacking PON1 are more susceptible
to organophosphate toxicity and are also more susceptible to
atherosclerosis when fed a high fat diet (7). Additionally PON1 has two
common phenotypes that affect catalysis with certain substrates, and
one phenotype correlates with a propensity toward vascular disease
(14). Additionally, there are polymorphisms that affect circulating
PON1 levels (15) that may alter the propensity to develop vascular disease.
Despite the association of PON1 with less vascular disease, two
essential components in defining a biologic role for PON1 remain
mysterious. The biologic substrate of PON1 is unknown, because it did
not evolve to detoxify organophosphorous pesticides and neurotoxins
and, at least in part because of the uncertainty over its substrates,
the way by which PON1 lessens atherogenesis is also unknown. The search
for a mechanistic role for PON1 reveals that it acts as an antioxidant
(11) and has peroxidase activity (6). However, the antioxidant activity
of PON1 is independent of its esterase activity (16), is at variance
with its aryl esterase activity in isoforms arising from genetic
polymorphisms (11, 16), and is unaffected by either heat or EDTA
treatment that destroy aryl esterase activity (16). PON1 has a single unpaired cysteine residue that mutagenesis shows has no role in catalysis or stability (17), but this residue is absolutely essential
for the antioxidant effect of PON1 (10, 18). The possibility that PON1
has two active sites that separately catalyze its esterase and
antioxidant activities has been raised (11).
Oxidized lipoprotein particles contain pro-inflammatory lipid mediators
that include ones that functionally mimic the potent phospholipid
mediator platelet-activating factor (PAF) (19). PAF initiates
physiologic inflammation and is active at picomolar levels. PAF is
recognized by a single molecularly characterized receptor found on a
number of cells that comprise the innate immune system (20). High
affinity recognition of PAF by the PAF receptor depends on an
sn-1 ether bond, the short acetyl sn-2 residue, and the choline head group of this phospholipid mediator. Oxidation of
sn-2 polyunsaturated fatty acyl residues of ether
phosphatidylcholines, as occurs during oxidation of lipoprotein
particles, generates phospholipid products with shortened
sn-2 residues that are inflammatory agents (21) because they
are PAF receptor agonists (22, 23). The oxidative formation of PAF-like
lipids results from an unregulated chemical reaction, in contrast to
the tight regulation of PAF synthesis (24), and this process occurs in
atherosclerotic lesions (25) and in the circulation after exposure to
cigarette smoke (26, 27).
Oxidatively fragmented phospholipids, like PAF, are substrates for PAF
acetylhydrolase (28, 29), a phospholipase A2 that only
hydrolyzes phospholipids with sn-2 residues that are short or contain an oxy function introduced during oxidative fragmentation. This enzyme is a serine esterase with the signature catalytic triad
(30), and so is sensitive to serine esterase inhibitors but not
divalent ion chelators. PAF acetylhydrolase circulates in association
with LDL and HDL particles and can migrate between them in a
pH-dependent fashion (31).
PON1 is reported to hydrolyze PAF (32) and oxidatively fragmented
phospholipids (5, 9, 12, 13), suggesting a potential mechanism that
could account for the vascular protective effect of PON1. The existence
of two distinct enzymes in the same lipoprotein particle apparently
catalyzing the same reaction has yet to be tested directly. Here, we
show that trace amounts of PAF acetylhydrolase contaminate PON1
preparations and that PON1 lacking PAF acetylhydrolase displays no
phospholipase activity.
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MATERIALS AND METHODS |
Cibacron blue 3GA-agarose (type 3000-CL), DEAE-Sepharose 6B,
concanavalin A-Sepharose 4B, deoxycholate,
methyl-
-D-mannopyranoside, IGEPAL CA-630, potassium
bromide, phenyl acetate, phospholipase A2 (bee venom),
phospholipase C (Bacillus cereus), and phospholipase D
(cabbage) were from Sigma, and Extracti-gel was from Pierce. Octadecylsilica cartridges, aminopropyl columns, and high pressure liquid chromatography grade solvents were from JT Baker Inc.
(Phillipsburg, NJ), and Pefabloc was from Pentapharm AG
(Basel, Switzerland). Human serum albumin was the product of Baxter
Healthcare Corp. (Glendale, CA), whereas Hank's balanced salt solution
was from BioWhittaker (Walkersville, MD). PAF, carbamyl-PAF,
2-O-methyl PAF, and the PAF receptor antagonist BN52021 were
from BioMol Research Laboratories, Inc. (Plymouth Meeting, PA).
Fura-2/AM was from Molecular Probes (Eugene, OR). ECL kits were from
Amersham Biosciences, and polyclonal anti-PAF acetylhydrolase antiserum and cognate blocking peptide were from Cayman Chemical Co. (Ann Arbor,
MI). Recombinant human PAF acetylhydrolase was from ICOS Corp.
(Bothell, WA). [3H]PAF (10 Ci/mmol) was the product of
American Radiolabeled Chemicals, Inc. (St. Louis, MO). PAF
acetylhydrolase-deficient plasma (0.078 units of PAF acetylhydrolase/ml
versus 1.5 units/ml from a healthy control) was a kind gift
from Drs. Kei Satoh and Tada-atsu Imaizumi (Hirosaki University,
Hirosaki, Japan). We prepared oxidized phospholipids from low density
lipoprotein isolated from normolipidic subjects as described (22,
23).
PON1 was purified from healthy human volunteers essentially as
described by Gan et al. (33) with minor modifications. Human blood was collected in heparin, and plasma was generated from this and
then calcified with CaCl2 to a final concentration of 1 mM before the resulting fibrin clot was separated by
centrifugation. In few experiments the plasma pH was adjusted to either
to pH 6.0 or to pH 9.0 with 1 N HCl or NaOH prior to an
overnight incubation. These plasma samples (9 ml) were adjusted to a
density of 1.3 with potassium bromide and then layered with 27 ml of
saline in a centrifuge tube prior to ultracentrifugation for 3 h
at 150,000 × g to separate HDL and LDL. HDL particles
enriched with PON1 were mixed with Cibacron blue 3GA-agarose in 50 mM Tris-HCl buffer (pH 8.0), and loosely bound proteins
were removed with 4 M NaCl buffer. PON1 bound to the
Cibacron blue was then eluted with 0.1% deoxycholate and mixed with
either DEAE-biogel or DEAE-Sepharose 6B in 0.1% IGEPAL CA-630 (rather
than Nonidet P-40) before PON1 with was eluted with a linear gradient
of NaCl (0.0-0.5 M) in 0.1% IGEPAL CA-630. DEAE fractions
containing aryl esterase activity were diluted and rechromatographed on
a DEAE-Sepharose 6B column. The final purification step used a
concanavalin A-Sepharose column, and material from this step was
concentrated with a Centricon apparatus. We resolved the proteins in
the various fractions by 12% SDS-PAGE. PON1 was made detergent-free by
passing it over Extracti-gel when the products of its
reaction(s) were to be analyzed in a bioassay.
Aryl esterase activity of PON1 was determined spectrophotometrically
using phenyl acetate as the substrate (2). The assay contained 1 mM phenyl acetate, 20 mM Tris-Cl (pH 8.0), and
1 mM CaCl2. Blanks without enzyme were used to
correct for spontaneous hydrolysis. Activity was calculated from the
molar extinction coefficient at 270 nm (using differences in the
absorbance of phenol versus phenyl acetate) of 1310 M
1 cm
1. One unit of aryl
esterase activity was defined as a micromole of phenyl acetate
hydrolyzed per min.
We measured PAF acetylhydrolase activity using
[3H]acetate-PAF as described by Stafforini et
al. (31, 34) and also by bioassay. For this, human neutrophils
were isolated by dextran sedimentation and centrifugation over Ficoll
as before (35). Neutrophils (2.25 × 106/ml) were
labeled with Fura-2/AM as before (23), and changes in intracellular
calcium concentration were measured by dual excitation at 340 and 380 nm with emission collected at 510 nm. The amount of PAF stated in the
figures (typically at 10
10 M) was pretreated
with detergent-free PON1 or recombinant PAF acetylhydrolase for the
specified time at 37 °C before the entire reaction was tested in the
Ca2+ mobilization assay. We find as little as 5 to 10 ng of
PAF acetylhydrolase in 15 min abolishes the calcium signal generated by
0.1 nM PAF. In some experiments, purified PON1 was
pretreated for 1 h at 37 °C with either EDTA (100 µM) or the serine esterase inhibitor Pefabloc (100 µM) prior to assay. In some experiments PAF or
2-O-methyl PAF were treated with 10 µg of bee venom
phospholipase A2 (in 0.5% human serum albumin in Hank's
balanced salt solution with 10 mM Ca2+), 10 units of phospholipase C (B. cereus), or 10 units of
phospholipase D (cabbage) for 2 h at 37 °C and then tested in
the Ca2+-flux bioassay. The analysis of Ca2+
flux in PMN was as described (23, 36).
We probed for PAF acetylhydrolase by immunoblotting after
electrophoretic separation by SDS-PAGE. The resolved proteins were transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA)
and probed with PAF acetylhydrolase polyclonal antibody (diluted 1:1000). A standard of truncated, non-glycosylated (the form approved for clinical trials) recombinant PAF acetylhydrolase was examined in a
separate lane of the gel. Peroxidase-conjugated goat anti-rabbit IgG
antibody was the secondary antibody (diluted 1:10000) with enhanced
chemiluminescence reagent used for visualization. In some experiments,
the primary anti-PAF acetylhydrolase antibody was pre-incubated with
200 µg per blot of the cognate blocking peptide.
 |
RESULTS |
Standard Preparations of Human PON1 Hydrolyze PAF and the Oxidized
Phospholipids of Oxidized LDL--
We purified PON1 as described (2,
33) and assayed its hydrolysis of PAF or the PAF-like phospholipids
produced during LDL oxidation (19, 22, 23). We chose a bioassay to
examine hydrolysis because inactivation of these lipid mediators is the important outcome, and because the extreme potency of these mediators makes assays other than biologic ones impractical. Here we loaded freshly isolated human PMN with the Ca2+-sensitive dye
Fura-2 and followed increases in intracellular Ca2+
by increases in the fluorescent ratio of the dye. We found that PMN
were maximally stimulated by 10
10 M synthetic
PAF (Fig. 1A) and by the
PAF-like phospholipids created during LDL oxidation (Fig.
1B). PAF receptor antagonists abolished both responses (not
shown) (23). Pre-incubation of either PAF (Fig. 1C) or the
oxidized phospholipids isolated from oxidized LDL (Fig. 1D)
with purified PON1 abolished the ability of these lipids to simulate
PMN. Heat inactivation of PON-1 (Fig. 1E) destroyed the
effectiveness of the preparation in degrading PAF. The positive control, recombinant PAF acetylhydrolase destroyed the biologic activity of PAF as expected (Fig. 1F). PON1 therefore
displays the reported (5, 32) anti-inflammatory activity and
inactivates PAF and PAF-like phospholipids generated during LDL
oxidation.

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Fig. 1.
Purified human PON1 and recombinant PAF
acetylhydrolase inactivate PAF and PAF-like oxidized
phospholipids. Freshly isolated human PMN were loaded with the
Ca2+-sensitive dye Fura-2, and increases in
intracellular Ca2+ levels were analyzed through the
340/380-nm emission ratio. The cells were treated (closed
arrow) with PAF (A) or oxidized phospholipids extracted
from oxidized LDL (B). Pre-treating PAF (C) or
oxidized phospholipids (D) with PON1 (20 units), but not
heat-inactivated PON1 (E), or recombinant PAF
acetylhydrolase (F) for 2 h inactivated the agonist and
prevented the stimulated increase in intracellular Ca2+.
The effect of PON1 or PAF acetylhydrolase was on the added substrate
and not on the Fura-2 PMN reporter cells, because the subsequent
addition of 0.1 nM PAF to the cells evoked a full response
(open arrows).
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Phospholipid Hydrolysis by PON1 Preparations Occurs at the sn-2
Position--
PON1 is a phosphotriesterase (37) and might be expected
to inactive phospholipid mediators by hydrolysis of the phosphodiester bonds of the phosphocholine head group. That is, its enzymatic mechanism would suggest that it functions as either a phospholipase C
or phospholipase D. We tested this postulate in the following way. The
PAF receptor is stimulated by PAF (Fig.
2A) but also by an
sn-2 ether analog of PAF, 2-O-methyl PAF. This
analog cannot be hydrolyzed at the sn-2 position by
esterases, yet remains susceptible to the actions of phospholipases C
and D. We first showed that phospholipase D (Fig. 2B) and
phospholipase C (Fig. 2C) were just as effective as
recombinant PAF acetylhydrolase, phospholipase A2, and PON1
(Fig. 2, D-F) in hydrolyzing and inactivating PAF. We
performed a parallel experiment with 2-O-methyl PAF to find that it was an active agonist (Fig. 2G) and that
phospholipase D (Fig. 2H) and phospholipase C (Fig.
2I) effectively inactivated this PAF analog. We found as
expected that neither recombinant PAF acetylhydrolase (Fig.
2J) nor phospholipase A2 (Fig. 2K)
hydrolyzed and inactivated this phospholipid. However, we also found
that purified PON1 did not inactivate this PAF analog (Fig.
2L). Additionally, we found that a second non-hydrolyzable
PAF analog, 2-carbamoyl-PAF, also was impervious to treatment with PON1
(not shown). The inference from this series of experiments is that PON1
functions as an sn-2-directed esterase and does not attack
phosphodiester bonds to inactivate PAF or related phospholipids.

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Fig. 2.
Purified human PON1 displays
phospholipase A2 but not phospholipase C or D
activity. A, PAF induces a Ca2+ flux in PMN
that is destroyed by phospholipase D (PLD; B),
phospholipase C (PLC; C), recombinant PAF
acetylhydrolase (rPAFAH; D), phospholipase A2
(PLA2; E), and purified human PON1
(F). In contrast, the Ca2+ flux induced by
2-O-methyl-PAF (2-O-Me-PAF; G), a PAF
analog with a non-hydrolyzable sn-2 methyl function, is
inactivated by phospholipase D (H) and phospholipase C
(I), but not by PAF acetylhydrolase (J),
phospholipase A2 (K), or PON1
(L).
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Hydrolysis of PAF by Purified PON1 Requires an Activated Serine
Residue--
PON1 is an EDTA-sensitive phosphotriesterase, yet the
above data showed it to function as an sn-2-directed
esterase. We therefore determined whether the serine esterase inhibitor
Pefabloc, which reacts with activated serine residues of enzymes such
as phospholipases A2 and PAF acetylhydrolase, would
suppress PON1 phospholipid hydrolysis. We found that Pefabloc
effectively suppressed hydrolysis and inactivation of PAF by PON1 (Fig.
3C). In contrast, EDTA, which
completely destabilizes and inactivates PON1 (2, 37), had no effect on
PAF hydrolysis by PON1 preparations (Fig. 3D). As a control for the effectiveness of EDTA, we measured the aryl esterase activity of PON1. We found that the pattern of EDTA and Pefabloc effects on this
activity of PON1 was the complete reverse of that for PAF hydrolysis;
EDTA destroyed PON1 aryl esterase activity, whereas Pefabloc was
without effect on this reaction (Fig. 3E).

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Fig. 3.
The serine esterase inhibitor Pefabloc, but
not EDTA, inhibits PAF hydrolysis by PON1. PAF (10 10
M) was treated with purified human PON1 (25-35 units) or
PON1 pretreated with 100 µM of the serine esterase
inhibitor Pefabloc or 100 µM EDTA for 60 min. The effect
of PAF (A) or PAF treated with PON1 (B), PAF
treated with Pefabloc-inhibited PON1 (C), or PAF after
incubation with EDTA-treated PON1 on FURA2-loaded PMN (D)
was determined as in Fig. 1. E, the effect of Pefabloc or
EDTA on the aryl esterase activity of PON1 was determined as stated
under "Materials and Methods." F, Western blot for PAF
acetylhydrolase in PON1-containing fractions eluting from the Cibacron
blue-agarose (cibacron), second DEAE-Sepharose
(DEAE), or concanavalin A-Sepharose (Con-A)
columns. Each lane contained 75-100 µg of protein.
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This outcome was unexpected should the anti-atherogenic effect of PON1
reside in its hydrolysis of PAF and oxidized phospholipids. We
considered the possibility that PAF acetylhydrolase, which is also a
constituent of certain HDL particles (31), might contaminate PON1
preparations. We tested for the presence of PAF acetylhydrolase in PON1
preparations of increasing purity by Western blotting. We found (Fig.
3F) that PON1 fractions isolated from Cibacron columns
(which very effectively bind PAF acetylhydrolase (34, 38)) still
contained detectable amounts of immunoreactive PAF acetylhydrolase.
Fractions eluting from the DEAE column containing PON1 still showed
significant amounts of PAF acetylhydrolase contamination, whereas the
final concanavalin A step of the PON1 purification scheme suppressed
PAF acetylhydrolase contamination as reported (2, 6). What we cannot be
sure of, however, is to what extent Western blotting detects PAF
acetylhydrolase contamination at levels sufficient to confer
phospholipase A2 activity to purified PON1.
PAF Hydrolytic Activity, but Not PON1, Migrates from HDL to LDL in
a pH-sensitive Fashion--
PAF acetylhydrolase has been reported to
be difficult to separate from PON1 (2, 6), and both are components of
HDL particles. PAF acetylhydrolase is also associated with LDL
particles, and its distribution between these lipoprotein species is
modulated by pH. We determined whether the distribution of PON1 aryl
esterase activity among lipoprotein particles was affected by pH in
parallel with shifts in PAF hydrolytic activity. The data in Fig.
4 show that aryl esterase activity is
exclusively found in fractions corresponding to HDL particles at either
acidic (Fig. 4A) or basic pH (Fig. 4B). In
contrast PAF hydrolysis is largely found at a density corresponding to
HDL under acidic conditions, whereas under basic conditions the
preponderance of PAF hydrolytic activity is found at the density of
LDL. This shift of PAF hydrolytic activity corresponds to the
distribution of immunoreactive PAF acetylhydrolase protein (Fig
4C), as expected (31).

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Fig. 4.
PAF acetylhydrolase and PAF hydrolytic
activity, but not PON1, redistribute between LDL and HDL fractions in a
pH-dependent fashion. Distribution of PAF
acetylhydrolase (solid lines) or aryl esterase (dashed
lines) activity in a density gradient after an overnight
incubation at an acidic (A) or basic (B) pH as
described under "Materials and Methods" is shown. LDL elutes in
fractions 18 to 25, whereas HDL is recovered in fractions 6 to 12. C, immunoblot of PAF acetylhydrolase of the stated amount of
recombinant human PAF acetylhydrolase or LDL and HDL fractions (100 µg) recovered from the density gradient in the above panels.
Glycosylated plasma PAF acetylhydrolase migrates as a mixture of
molecular weights and migrates more slowly than truncated,
unglycosylated recombinant PAF acetylhydrolase.
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Detection of PAF Acetylhydrolase by Immunoblotting Is Not
Sufficiently Sensitive to Detect PAF Acetylhydrolase
Contamination--
We noted from the Western blot in Fig.
4C that we could detect 100 ng of the recombinant PAF
acetylhydrolase standard but could not reliably detect 10 ng of the
enzyme. PAF acetylhydrolase at levels less than 10 ng would therefore
be undetectable by immunoblotting and might be an invisible contaminant
of PON1 prepared from HDL, a source of both activities. We determined
the minimum amount of PAF acetylhydrolase that would be sufficient to
inactivate PAF in the Ca2+ flux bioassay by testing a
graded amount of recombinant enzyme on the 0.1 nM PAF we
use as an agonist in these assays. We found (Fig.
5A) that 800 pg or 1 ng of PAF
acetylhydrolase did not diminish the effectiveness of PAF in the
bioassay but that 5 ng of the enzyme noticeably reduced the amount of
available PAF. We also found that 10 ng completely suppressed the
PAF-induced Ca2+ response of PMN. We also varied the amount
of PAF acetylhydrolase in a radiometric assay of
[3H]acetyl-PAF hydrolysis and confirmed that 10 ng of the
recombinant enzyme is sufficient to inactivate half of the added
substrate (Fig. 5B) in just 15 min. Assays of PON1
inactivation of oxidized phospholipids may extend for many hours (5),
so trace amounts of PAF acetylhydrolase may be problematic.

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Fig. 5.
The amount of PAF acetylhydrolase sufficient
to rapidly inactivate PAF is less than can be detected by Western
blotting. A, PAF (10 10 M) was
treated with the stated concentration of recombinant PAF
acetylhydrolase and then assayed as a Ca2+-mobilizing
agonist for FURA-2-loaded PMN as in Fig. 1. B,
[3H]PAF hydrolysis as a function of PAF acetylhydrolase
concentration. Hydrolysis of [3H]PAF in 15 min by the
stated amount of PAF acetylhydrolase was determined as stated under
"Materials and Methods."
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PON1 Can Be Separated from PAF Acetylhydrolase--
We purified
PON1 to apparent homogeneity (Fig.
6A) and a final aryl esterase
specific activity of 238-400 units/mg. We noted that different
preparations of PON1 expressed a variable ratio of PAF hydrolysis to
aryl esterase activity (not shown), an observation consistent with PAF
acetylhydrolase co-purifying with PON1 (2, 6). We reasoned that if we
started the purification of PON1 from a source that contained less PAF
acetylhydrolase, that at a minimum we would garner evidence for PAF
acetylhydrolase contamination of purified PON1 or that we may succeed
in completely separating PAF acetylhydrolase from PON1. We
pre-incubated plasma overnight at an alkaline pH to deplete PAF
acetylhydrolase content of HDL, isolated this HDL, and used this as a
starting source for PON1 purification. We examined fractions obtained
from neutral plasma as the starting source to find that there were
detectable amounts of PAF acetylhydrolase in the first two steps of the
purification (Fig. 6B), but that even the first step of PON1
purification from PAF acetylhydrolase-depleted HDL was free of this
contaminating enzyme by immunoblot analysis.

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Fig. 6.
PON1 purified from HDL depleted of PAF
acetylhydrolase does not act as a phospholipase. A,
Coomassie Blue-stained SDS-PAGE of fractions obtained during PON1
purification from PAF acetylhydrolase-depleted HDL. Lane 1,
molecular mass markers; lane 2, Cibacron fraction;
lane 3, DEAE fractions 39-45; lane 4, DEAE
fractions 46-52; lane 5, DEAE fractions 28-34; lane
6, second DEAE column fractions 39-45; lane 7, PON1
control; lane 8, Concanavalin A fraction. B,
Western blot for PAF acetylhydrolase in PON1 fractions isolated from
neutral plasma or from HDL isolated from plasma incubated overnight at
an alkaline pH to shift PAF acetylhydrolase out of HDL. C,
effect of PON1 purified in two ways on PAF or oxidized phospholipids
extracted from oxidized LDL. PON1 was purified from plasma at a neutral
pH or from HDL depleted of PAF acetylhydrolase as above and tested for
the ability to inactivate PAF as an agonist for Fura-2 loaded
PMN as described in Fig. 1.
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However the above results show that even when PAF acetylhydrolase
cannot be detected by an immunoblot, there still may be enough enzyme
to inactivate significant amounts PAF. We then compared PAF hydrolysis
by the PON1 preparation apparently free of PAF acetylhydrolase to
material purified in the standard way from neutral plasma, which had
the same specific final aryl esterase activity. We found that this
strategy was effective in separating aryl esterase activity from PAF
hydrolytic activity, because PON1 purified from HDL depleted of PAF
acetylhydrolase by pre-incubation at an alkaline pH did not hydrolyze
PAF (Fig. 6C), whereas PON1 purified using a standard
protocol hydrolyzed PAF as before. We tested the PON1 preparation
devoid of PAF hydrolytic activity for its ability to inactive the
PAF-like lipids found in oxidized LDL. PON1 devoid of PAF
acetylhydrolase, in contrast to a standard preparation of PON1, also
lacked oxidized phospholipid phospholipase activity (Fig.
6C). Therefore PON1 does not, by itself, hydrolyze either
PAF or oxidized phospholipids possessing PAF-like bioactivity.
Genetic Ablation of PAF Acetylhydrolase Shows PON1 Has No Role in
PAF or Oxidized Phospholipid Metabolism--
There are genetic
polymorphisms and mutations in the human PAF acetylhydrolase gene, and
the more common of these produces catalytically inactive PAF
acetylhydrolase that does not persist in the circulation (39). Plasma
from such an individual offers the opportunity to test the contribution
of PON1 to PAF catabolism. We found that plasma from an individual with
wild-type PAF acetylhydrolase completely inactivated PAF, whereas
plasma from an individual with a mutant form of the gene did not (Fig.
7A). This difference in PAF
hydrolytic capability did not arise from the presence of an inhibitory
agent as a mixing experiment showed that the plasma from the mutant
donor did not affect PAF hydrolysis by normal plasma or recombinant PAF
acetylhydrolase. We repeated this experiment (Fig.
7B) using oxidized phospholipids as the substrate with
similar results; a point mutation in the gene encoding PAF
acetylhydrolase abolishes the hydrolysis of oxidized phospholipids with
PAF-like bioactivity. We then measured aryl esterase activity in plasma from individuals with wild-type or mutant PAF acetylhydrolase to find
that there was no diminution in PON1 activity by this mutation (Fig.
7C). The bulk of the plasma aryl esterase activity in both
types of plasma was sensitive to EDTA and therefore reflects PON1
activity. Thus even though both PON1 and PAF acetylhydrolase are
components of HDL, their expression is independent of one another.

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Fig. 7.
Plasma from an individual with a mutation in
the PAF acetylhydrolase gene contains paraoxonase activity but does not
hydrolyze PAF. A, plasma from an individual with a
mutation in the PAF acetylhydrolase gene does not hydrolyze PAF, and
mixing experiments with recombinant PAF acetylhydrolase or normal
plasma show the lack of activity does not arise from the presence of an
inhibitor. B, plasma from an individual with a mutation in
the PAF acetylhydrolase gene does not hydrolyze oxidized phospholipids
extracted from oxidized LDL. Mixing experiments show the loss of this
activity is not because of an inhibitor. C, plasma from an
individual lacking PAF acetylhydrolase retains a normal level of PON1
aryl esterase activity. Aryl esterase activity was measured as
described under "Materials and Methods" with or without a
preincubation with 1 mM EDTA to destabilize and inhibit
PON1.
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DISCUSSION |
We discovered that highly purified preparations of human PON1
inactivate PAF and PAF-like oxidatively fragmented phospholipids by
hydrolyzing the sn-2 residue to produce two inactive
products, lysoPAF and a short chain free fatty acid. This reaction
suppresses atherogenesis, and it protects lipoprotein particles from
chemically reactive phospholipids. Genetic deletion of PON1 activity in
mice creates animals that are susceptible to organophosphate
intoxication and, importantly, demonstrate an increased propensity to
form atherosclerotic lesions when placed on a high fat diet or an apoE knock-out background (7, 40, 41). Conversely, transgenic mice with
increased levels of circulating PON1 are less susceptible to developing
atherosclerosis (42, 43), as are mice expressing PAF acetylhydrolase as
a transgene (44). These observations, coupled with extensive literature
(4-13) indicating that paraoxonase is beneficial and hydrolyzes
PAF-like phospholipid oxidation products that are formed during LDL
oxidation (19, 23) to inactive products, suggest that this is the way
PON1 exerts its salubrious effects. Hydrolysis of oxidatively modified
and fragmented phospholipids that are PAF receptor agonists (19, 23,
45), peroxisome proliferator-activated receptor ligands and
agonists (46), and structurally inappropriate and disruptive
phospholipids (47-50) are of unquestioned value in protecting against
atherosclerosis, but this reaction is not accomplished by PON1.
PON1 is an organophosphatase (51) that protects animals
that express it against intoxication by organophosphate insecticides (7). If PON1 attacks the phosphodiester of biologically active phosphatidylcholines then it would inactivate
1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine, a PAF analog that is not a substrate for the esterolytic activity of
phospholipases A1 or A2. It did not. Instead we
find that purified human PON1 inactivated PAF, as reported (32), where
the single bond susceptible to esterolytic activity resides at the
sn-2 position. This is the same type of activity displayed
by the PAF acetylhydrolase that is also a component of certain HDL
particles (31).
Physical resolution of PON1 and PAF acetylhydrolase has been found
previously to be challenging (2, 6, 32). We, like other investigators
(32), tested our PON1 preparation for the presence of PAF
acetylhydrolase by Western blotting for the enzyme with negative
results. Other investigators have searched for PAF acetylhydrolase
contamination by mass spectroscopy (12, 13) and sequencing (32). But
whether any of these methods is sufficient to detect PAF
acetylhydrolase contamination at low levels, but levels still
sufficient to account for the observed catalytic activity, have not
been investigated. We find that we easily detect 100 ng, but not 10 ng,
of PAF acetylhydrolase by Western blotting. We also find, however, that
as little as 5 to 10 ng of PAF acetylhydrolase is sufficient to account
for all of the phospholipase activity in our purified PON1 preparations.
Could low levels of PAF acetylhydrolase confer phospholipase activity
to purified PON1? Certainly this is of concern when prolonged
incubations are employed (5) when we find a few nanograms of PAF
acetylhydrolase destroys PAF and oxidized phospholipids in a little as
15 min. We find that hydrolysis of PAF and PAF-like oxidized
phospholipids by purified human PON1 was insensitive to EDTA, yet PON1,
and especially human PON1 (2), is not stable in the absence of
Ca2+ and other divalent cations. Human PON1 is even
inactivated by choosing EDTA over heparin during a blood draw (3). We
find (34) that PAF acetylhydrolase is a Ca2+-independent
phospholipase A2 that is insensitive to EDTA.
Conversely, PAF acetylhydrolase with its classic serine esterase
GXSXG motif is inhibited by compounds like
Pefabloc (52) that derivatize activated serines. Pefabloc effectively
inhibited PAF and oxidized phospholipid hydrolysis by purified PON1,
but PON1 does not contain a signature serine esterase motif (53). Rather, histidine, aspartate, glutamate, and a tryptophan residues are
essential for paraoxonase activity (54). PON1 is not inhibited by
serine esterase inhibitors like diisopropylfluorophosphate (37), which
in fact is a substrate (33, 55). Experiments with inhibitors indicate
that there is a potential for PAF acetylhydrolase to contaminate and
alter the apparent enzymatic activity of PON1. This conclusion is
strengthened by the report that PON1 knock-out mice show no diminution
in their levels of PAF acetylhydrolase activity (7).
PON1 and PAF acetylhydrolase are localized in the same lipoprotein
particle, have the same molecular weights (33, 34), and share physical
properties that make complete separation difficult. There is a
distinguishing property of PAF acetylhydrolase that has proved useful
in separating these two enzymes. Two-thirds of serum PAF
acetylhydrolase is associated with LDL particles, and one-third is
associated with apoE-containing HDL particles (31), but this
distribution can be manipulated. A brief incubation at alkaline pH
transfers HDL-associated PAF acetylhydrolase activity to the LDL
fraction, and the converse result is obtained under acidic conditions
(31). We found this maneuver caused PAF acetylhydrolase mass and
activity to redistribute to the LDL fraction leaving PON1 in HDL. This
result offers a further indirect indication that PON1 does not
significantly contribute to PAF hydrolysis, because PAF hydrolytic
activity tracked with PAF acetylhydrolase mass.
PON1 purified from PAF acetylhydrolase-depleted HDL, in contrast to
that purified in the standard way from plasma, did not show
phospholipase A2-like activity and did not hydrolyze PAF. The PON1 preparation free of PAF hydrolytic activity also did not
inactivate PAF-like oxidized phospholipids. Physical separation of the
two enzymes allows us to conclude that PON1 is not an oxidized phospholipid phospholipase. In fact PAF acetylhydrolase is the only
lipoprotein-associated enzyme with this activity, as shown by the
complete lack of PAF and oxidized phospholipid hydrolytic activity in
the plasma of an individual with an inactivating mutation of the PAF
acetylhydrolase gene. Plasma from this individual displayed full aryl
esterase activity, so these two activities are also genetically
separable. The active site responsible for the aryl esterase activity
of PON1 is clearly separable from that catalyzing PAF hydrolysis (32),
because two enzymes are responsible for the two unrelated activities.
We greatly appreciate the gift of PAF
acetylhydrolase-deficient plasma from Tada-atsu Imaizumi and Kei Satoh
(Hirosaki University). We thank Jason Hansen for early experiments
assaying paraoxonase activity, Dee Biddle for Western analysis,
Stephen Prescott for thoughtful input, and Cletus D'Souza
for initial experiments, and we appreciate the technical aid of Donnie
Benson, Jennifer Eyre, and Margaret Vogel. We also appreciate the
graphics created by Diana Lim.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M211126200
The abbreviations used are:
PON1, paraoxonase-1;
HDL, high density lipoprotein;
LDL, low density
lipoprotein;
PAF, platelet-activating factor;
PMN, polymorphonuclear
leukocyte.
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