From the Program in Human Molecular Biology and
Genetics,
Huntsman Cancer Institute, and
¶ Cardiovascular Research and Training Institute, University of
Utah, Salt Lake City, Utah 84112
Platelet-activating factor acetylhydrolases are
structurally diverse isoenzymes that catalyze the hydrolysis of the
acyl group at the second position of glycerol in unusual, bioactive
phospholipids (Fig. 1). Thus, as categorized by
enzymatic activity, they are phospholipases A2 (Groups VII
and VIII, Ref. 1), which often initiate signal transduction and are
regulated by the state of the cell activation. However, the
platelet-activating factor (PAF,1
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
acetylhydrolases have the opposite role; they were discovered by
investigators who focused on the inactivation of PAF (2). The
phospholipid PAF has diverse physiological and pathological functions,
and its inactivation was identified as an important step in regulating the overall biological function; indeed PAF acetylhydrolase has been
termed a signal terminator (3). However, an immediate problem is
apparent; phospholipases A2 are a common component of
venoms, and their role in this context is to facilitate the spread of
toxins by degrading membrane phospholipids; thus, how could there be
such an active enzyme circulating in blood and present in the cytoplasm
of cells? The answer came when it was shown that the PAF
acetylhydrolases have marked selectivity for phospholipids with short
acyl chains at the sn-2 position; with chains longer than
nine carbons there was essentially no measurable activity (Fig.
2) (4, 5). Thus, normal membrane phospholipids are
protected from hydrolysis by an intrinsic property of the enzyme.
Subsequently, it was discovered that certain forms of PAF
acetylhydrolases have a broader spectrum of action; they hydrolyze phospholipids containing relatively long sn-2 acyl chains
(up to 9 methylene groups, Ref. 5). However, in this case, the suitability of a phospholipid as a substrate depends on another form of
unusual sn-2 acyl group, one that contains a carbonyl group
at the
The initial studies on PAF acetylhydrolases were performed on the
secreted form found in mammalian plasma; this isoform circulates in
blood as a complex with lipoproteins (6, 7). In addition, PAF
acetylhydrolase activities were identified in the cytosolic fraction of
various mammalian tissues (8, 9) and human blood cells (9, 10). These
activities have substrate specificities quite similar to that of the
plasma PAF acetylhydrolase and thus belong to the same group of
calcium-independent phospholipases A2. However, the plasma
(11) and intracellular (12-14) forms of PAF acetylhydrolase are
encoded by individual genes, and the identity among these varies
widely, depending on the intracellular isoform. It is not yet precisely
known how each domain affects the function of each PAF acetylhydrolase,
but the diversity in gene structure most likely serves to determine
specific roles played by each member of the group.
PAF, the phospholipid that led to the discovery of PAF acetylhydrolase,
is a mediator of a wide range of immune and allergic reactions (15,
16). It activates inflammatory cells at very low concentrations
(10 Plasma (Secreted) PAF Acetylhydrolase The cloning of a cDNA encoding the plasma form of PAF
acetylhydrolase was reported in 1995 (11) and subsequently was
confirmed by other groups (21, 22). The gene is expressed in thymus, tonsil, and placenta but not in heart, kidney, or cerebral cortex. However, there is expression of this form of PAF acetylhydrolase in
other areas of the brain (11). This is interesting since PAF has been
implicated as a physiological messenger in long term potentiation (23)
and, when present in excess, as a pathological mediator, for example in
seizures (24). However, whether the plasma form of PAF acetylhydrolase
is responsible for regulating the amount of PAF in the brain is not
clear since mRNAs for intracellular PAF acetylhydrolases occur in
specific brain regions as well (12, 13).
The cDNA for human plasma PAF acetylhydrolase encodes a protein
whose first 17 residues (Met-1 to Ala-17) are hydrophobic and
presumably target the protein for secretion. The next 24 predicted residues (Val-18 to Lys-41) were not found in the protein purified from
plasma, and it is not yet clear whether these represent a prepro-peptide or whether the protein isolated from human plasma had
undergone proteolytic degradation. The primary structure of plasma PAF
acetylhydrolase is unique and includes only a small region of homology,
a GXSXG motif found in serine esterases,
including many lipases. The active site of such esterases contains a
catalytic triad composed of a serine, an acidic residue (usually
aspartate), and a histidine residue. In plasma PAF acetylhydrolase, the
components of the active site triad have been identified by
site-directed mutagenesis; Ser-273, Asp-296, and His-351 are essential
for catalytic activity (25). From these observations it is likely that
the structures of the active sites of this enzyme and several neutral lipases are similar.
Several observations indicate that synthesis and secretion of the
plasma form of PAF acetylhydrolase are hormonally regulated in
vivo. Miyaura et al. (26) showed that estrogens
decrease secretion by macrophages and that progestins have the opposite effect. These studies were complemented by the observation that the PAF
acetylhydrolase activity in maternal plasma decreased dramatically
before parturition (27) and returned to basal levels soon afterward.
Based on these findings, Johnston and colleagues (27) proposed that
during early pregnancy the high PAF acetylhydrolase levels ensure that
the concentrations of PAF will be low; this protects against premature
uterine contraction since PAF is a potent agonist for this response. In
contrast, the fall in enzymatic activity late in pregnancy may allow
PAF to accumulate and initiate parturition. The conclusion that
estrogen has a regulatory role is supported by population studies in
which it has been found that women have a lower level of plasma PAF
acetylhydrolase activity than men (28).2 In
addition to the sex hormone regulation, dexamethasone, an anti-inflammatory glucocorticoid, was found to increase PAF
acetylhydrolase levels in the plasma of rats (29). Another example of
regulation of this activity is developmental; at birth the levels are
very low, but there is a marked increase during the first weeks of life
(30).2 The basis for this is unknown. In adults, there is a
strong correlation between the plasma activity and the cholesterol or
low density lipoprotein (LDL) level (31-33); again the basis is
undetermined. This may be important since the enzyme circulates bound
to lipoproteins and the fraction associated with LDL seems to be more
active under some conditions (34). This may result from an allosteric
effect of the binding or may represent an effect of the lipoprotein
environment on substrate availability. Finally, Satoh et al.
(35) showed that PAF itself can induce the synthesis and secretion of
the plasma PAF acetylhydrolase, a response that may indicate a form of
feedback regulation that serves to protect the organism against excessive signaling by PAF and related lipids.
The plasma form of PAF acetylhydrolase has been the target of many
studies testing its association with inflammatory diseases (reviewed in
Ref. 36). Modest changes in plasma activity have been described in
asthma, hypertension, vascular disease, atherosclerosis, sepsis,
necrotizing enterocolitis, and others (36). In most cases, the activity
increased during the acute phase, suggesting that this may be a
physiological response to inflammatory stimuli, perhaps PAF itself as
described above. A limiting feature of these studies is that they were
restricted to measurements of enzymatic activity since antibodies have
only recently become available. This may be important since several
groups have shown that the plasma PAF acetylhydrolase is inactivated by
oxidants and many of the disorders studied would include oxidant and/or
free radical formation (37). Paradoxically, the most important
substrates of this enzyme are the products of oxidative reactions, and
these compounds inactivate the enzyme themselves. Thus, although
informative, studies that determine changes in the overall levels of
PAF acetylhydrolase activity are difficult to interpret precisely
because the final level of activity is the result of diverse factors
with (sometimes) opposite effects. Animal studies using recombinant PAF
acetylhydrolase have provided the best clues into the role of this
enzyme in disease; pretreatment with recombinant PAF acetylhydrolase
blocks PAF-induced edema (11), prevents asthma-related symptoms, and
protects animals from septicemia.2 Thus, this enzyme
prevents not only inflammatory conditions induced by administration of
exogenous PAF but also diseases in which the production of PAF and/or
oxidized phospholipids is suspected to occur.
An important medical area in which plasma PAF acetylhydrolase may play
a protective role is in oxidative processes that are thought to be a
component of early vascular disease. The phospholipids in LDL particles
are known to undergo oxidation under experimental conditions, and
evidence for this in vivo has been presented as well (38).
The oxidized phospholipids are substrates for PAF acetylhydrolase, and
addition of the enzyme prevents the formation of minimally modified and
modified LDL in vitro (39, 40). The products of hydrolysis,
lysophosphatidylcholine and fragments of fatty acids, are water-soluble
and can be further metabolized to products that are not toxic. In
contrast, the phospholipids with an oxidized fatty acid, perhaps with
an aldehyde at the It has been known since 1988 that approximately 4% of the Japanese
population has undetectable levels of plasma PAF acetylhydrolase activity, and the prevalence is even higher in children with severe asthma (41). The molecular basis for the deficiency was discovered recently, and it results from a mutation that converts Val-279, which
is conserved in five species examined, to Phe (42). In Japan, this
mutation occurs as a heterozygous trait in 27% of the population,
which indicates that the population is in Hardy-Weinberg equilibrium
for this trait. Surprisingly, given the remarkable prevalence in Japan,
this mutation was not found in North American subjects and appears to
be restricted to Asia. Several groups are now testing whether this
mutation increases the susceptibility to inflammatory or allergic
diseases. One possibility is that the plasma PAF acetylhydrolase
functions normally as an anti-inflammatory safety net, much as do the
plasma anti-proteases. If this is true, then deficiency of the enzyme
should be a risk factor for developing relevant diseases (or enhancing
their severity), but the disease might be manifest only when the
mutation occurs in conjunction with other genetic predisposition or if
the individual encounters the appropriate environmental factors.
Intracellular PAF Acetylhydrolases In addition to the extracellular plasma enzyme, intracellular PAF
acetylhydrolase activities have been reported to regulate the
accumulation of PAF under some circumstances; for example, in
macrophages (43, 44) and platelets (45, 46) the level of PAF
accumulation is determined by the PAF acetylhydrolase activity. The
intracellular PAF acetylhydrolase activities present in brain, kidney,
and liver have been extensively characterized. Inoue's group described
enzymatic activities that they designated isoforms I and II, and
isoform I proved to have more than one enzyme so it was subdivided.
Subsequently, they have isolated three different cDNAs encoding
catalytically active, intracellular PAF acetylhydrolases (Table
I) (47). The minimal essential elements determining the
catalytic reaction per se are shared by all PAF
acetylhydrolases, including secreted and intracellular forms: the
GXSXG or GXSXV motif
characteristic of lipases and esterases (Table I) (47). Features such
as localization, structure of the protein, and substrate binding site
are likely to be defined by regions of the gene that differ widely
among isoforms.
Table I.
Secreted and intracellular PAF
acetylhydrolases
-end of the acyl chain (5). The markedly restricted substrate
specificity of PAF acetylhydrolases is unusual among phospholipases
A2, and the unifying feature of the substrates utilized is
that they have potent biological actions, which can lead to
pathological events when they accumulate inappropriately. The nature of
the substrates hydrolyzed by PAF acetylhydrolases points at key roles
for these activities in physiology and pathology, and it has provided
important clues into what is currently thought to be the main function
of these enzymes, which is to act as scavengers of bioactive
phospholipids.
Fig. 1.
Reaction catalyzed by PAF
acetylhydrolases. PAF acetylhydrolase recognizes PAF and also a
family of structurally related phospholipids, some of which contain
oxidized functionalities at the second position of the glycerol
backbone. The type of linkage at the sn-1 position does not
affect catalytic activity. The reaction products are lyso derivatives
of phospholipid substrates and short fatty acids.
[View Larger Version of this Image (11K GIF file)]
Fig. 2.
PAF acetylhydrolases have marked specificity
for PAF and structurally similar phospholipids. The ability of PAF
acetylhydrolases to recognize phospholipid substrates varies with each
isoform. The plasma (secreted) PAF acetylhydrolase and intracellular
isoform II recognize PAF and phospholipids with up to 9 methylene
groups at the sn-2 position of phospholipid substrates. In
addition, the suitability of a phospholipid increases if the
sn-2 acyl group has an oxidized functionality, such as an
aldehydic or carboxylic group. In contrast, intracellular isoform Ib,
which includes catalytically active subunits and
, is strictly
specific for PAF hydrolysis. These subunits do not recognize
phospholipids with medium length sn-2 chains, regardless of
the presence of oxidized functionalities, and their function is limited
to precisely regulate the levels of PAF. Orange bar,
extracellular PAF acetylhydrolase and intracellular isoform II;
white bar, intracellular PAF acetylhydrolase isoform Ib (
and
subunits); gold bar, oxidized functionality.
[View Larger Version of this Image (29K GIF file)]
10-10
12 M) through a
G-protein-linked, serpentine receptor, and it is synthesized in a
regulated pathway(s) in response to a variety of agonists. If the
mechanism for PAF inactivation is impaired in some way, the return to
basal conditions may be compromised, resulting in prolonged
inflammation and inappropriately long recruitment of effector cells to
sites of injury. The second group of compounds hydrolyzed by PAF
acetylhydrolases, the oxidatively fragmented phospholipids (17, 18),
also has short acyl groups at the sn-2 position of glycerol,
but they are derived from oxidation of polyunsaturated fatty acids that
occupy this position in the phospholipids of cellular membranes. These
compounds apparently mimic the structure of PAF closely enough to bind
to its receptor and thereby elicit the same responses. The similarities
between PAF and the oxidatively fragmented phospholipids are contrasted by one essential difference: the synthesis of PAF is highly controlled (16, 19, 20) whereas oxidized phospholipids are produced in an
unregulated manner. Therefore, the extent to which products of
phospholipid oxidation will accumulate depends very heavily on the rate
at which they are catabolized. The fact that PAF acetylhydrolases are
maximally active in the basal state and do not require calcium for
activity ensures that these activities provide an immediate defense
mechanism against toxic effects mediated by fragmented phospholipids.
-end, could be detrimental in two ways; these
phospholipids mimic PAF to support inflammation but, in addition, could
covalently modify the apolipoprotein. This results in unregulated
cholesterol ester accumulation by macrophages, which become the foam
cells characteristic of atherosclerosis. LDL-associated PAF
acetylhydrolase can prevent the modification of LDL to an atherogenic
particle (39), likely by the hydrolysis of reactive oxidatively
fragmented phospholipids before they can derivatize apoB-100 to a form
recognized by scavenger receptors. The lysophosphatidylcholine formed
in the PAF acetylhydrolase reaction also can provoke pathological
reactions in the cells of the vascular wall, but this effect occurs
only at concentrations at least 1000-fold higher than that required for
the effects of PAF and oxidized phospholipids. Thus, PAF
acetylhydrolase provides LDL particles with an intrinsic protective
mechanism that minimizes the damaging effects caused by lipid
peroxidation reactions.
Hattori et al. (14) have studied isoform II extensively and
recently reported the cloning of a cDNA encoding this enzyme. This
isoform, which is expressed in liver and kidney, has active site serine
and cysteine residues, and its substrate specificity is similar to that
of the secreted, plasma activity; isoform II catalyzes the hydrolysis
of phospholipids with acyl chains containing up to five methylene
groups. This suggests that one function of isoform II may be to
scavenge oxidatively fragmented phospholipids (48), much like the
plasma PAF acetylhydrolase. Interestingly, the amino acid sequence of
isoform II shows 41% identity with the plasma PAF acetylhydrolase
(Fig. 3A) (14). These results indicate that
the regions shared by these PAF acetylhydrolases include determinants
of substrate specificity and that the two activities may share a common
physiologic function.
The most thoroughly studied of the intracellular enzymes is isoform Ib,
which has three subunits of molecular masses 45, 30, and 29 kDa (,
, and
, respectively) (49). This isoform is entirely specific for
PAF hydrolysis, i.e. it does not recognize oxidized
phospholipids as substrates, which indicates that its function is to
precisely modulate PAF levels exclusively of other substrates (49). The
29-kDa subunit is catalytically active, exhibits no overall homology
with other proteins, and contains a modified version of the consensus
sequence of the serine esterase family that has been found in other PAF
acetylhydrolases (Table I). It has been crystallized and a high
resolution structure determined; a key finding was that the tertiary
fold of this protein is markedly similar to that found in
p21ras and other GTPases (50). In addition, the active site is
made of a trypsin-like triad of Ser-His-Asp, and its chirality is the same as that found in other esterases and neutral lipases. The 30-kDa
subunit is homologous (63.2% identity) with the 29-kDa subunit (Fig.
3B), and it contains the same catalytic triad present in the
29-kDa subunit (13). The 45-kDa subunit of isoform Ib has no catalytic
activity, and it has been proposed that it may regulate the activity,
location, or turnover of the holoenzyme (50). This subunit of isoform
Ib is one of the most conserved proteins known, and it has received
much attention because mutations in the human gene (LIS-1)
are responsible for Miller-Dieker lissencephaly (51), a devastating
neurological disease whose hallmark is severe brain malformation
manifested by a smooth cerebral surface (52). The cellular basis of the
disease appears to be abnormal neuronal migration during development
(52). The finding that a subunit of intracellular PAF acetylhydrolase
plays an important role in brain development may be related to the work
of Kato et al. (23), who concluded that the substrate, PAF,
is a retrograde messenger in long term potentiation. A current model
proposes that PAF acetylhydrolase serves to modulate PAF levels, which
can be toxic at high concentrations, during brain development (24) and
that hydrolysis of PAF may induce conformational changes in the
heterotrimeric PAF acetylhydrolase complex that affect the ability of
the 45-kDa subunit to interact with cytoskeletal proteins (50). Three
lines of evidence support the existence of PAF acetylhydrolase Ib as a
heterotrimer in vivo. First, the three subunits co-purify
throughout several chromatographic steps. Second the crystal structure
of the 29-kDa subunit is reminiscent of a G-protein-like heterotrimer.
Third, mRNAs for the three subunits are co-expressed in the
developing brain (53). Thus, it is likely that the heterotrimer is
representative of the structure that this isoform adopts in
vivo and that this complex serves to carefully modulate the levels
of PAF in the brain. The fact that this isoform has an even higher
specificity for PAF than the others suggests that it may have a
specific role in a signal transduction pathway, whereas the plasma
enzyme and intracellular isoform II may have a more fail-safe role,
i.e. to inactivate PAF and oxidized phospholipids that have
been generated in the wrong quantities or the wrong place and are
likely to cause damage.
The human erythrocyte PAF acetylhydrolase activity has been purified and shown to be composed of two identical 25-kDa subunits; it exhibits surface dilution kinetics and has biochemical properties that differentiate it from the plasma activity (54). However, this activity is an isozyme of the PAF acetylhydrolase family because it is calcium-independent and hydrolyzes phospholipids containing short and/or oxidized acyl groups at the sn-2 position. The enzyme is a serine esterase and requires reducing agents for maximal activity, much like isoform II. The most likely role of the erythrocyte PAF acetylhydrolase in vivo is the hydrolysis of the phospholipid products of oxidative fragmentation of membrane phospholipids. This step may serve to hinder further oxidative reactions and to reduce the toxic effects of oxidized lipids. Moreover, hydrolysis allows subsequent restoration of the membrane integrity by reacylation of the lyso derivatives with long chain fatty acyl groups, as can also occur in red blood cells (55).
Much has been learned about the PAF acetylhydrolases; the last 3 years have been particularly fruitful because the tools generated (cDNAs, recombinant proteins, and antibodies) will allow additional dissection at a molecular level. How are the PAF acetylhydrolase genes regulated? What is the role of each of the isoforms? Do they serve a general antioxidant role? What is the structural basis for the marked substrate specificity? What structural features determine the binding of the plasma PAF acetylhydrolase to specific lipoproteins in the blood? What functional benefit is conferred by the trimeric structure of the intracellular type Ib? In addition, the next few years will see the development of genetically engineered animals, as well as clinical studies of whether these enzymes have the postulated protective effects against inflammatory and allergic diseases.
We are grateful to the students and postdoctoral fellows who carried out studies in our laboratories and to our collaborators at ICOS Corporation (Bothell, WA), particularly Larry Tjoelker, Chris Eberhardt, and Pat Gray. In addition, the group of Professor Keizo Inoue (Tokyo) has been extraordinarily generous in sharing their results.