(Received for publication, July 26, 1995; and in revised form, August 23, 1995)
From the
Platelet-activating factor (PAF) is a potent pro-inflammatory
autacoid with diverse physiological and pathological actions. These
actions are modulated by PAF acetylhydrolase, which hydrolyzes the sn-2 ester bond to yield the biologically inactive lyso-PAF.
In contrast to most secreted phospholipase As, plasma PAF
acetylhydrolase is calcium-independent and contains a
GXSXG motif that is characteristic of the neutral
lipases and serine esterases. In this study we tested whether the
serine in this motif is part of the active site of plasma PAF
acetylhydrolase and, if so, what the other components of the active
site are. Using site-directed mutagenesis, we demonstrated that Ser-273
(of the GXSXG motif), Asp-296, and His-351 are
essential for catalysis. These residues were conserved in PAF
acetylhydrolase sequences isolated from bovine, dog, mouse, and
chicken. The linear orientation and spacing of these catalytic residues
are consistent with the
/
hydrolase conformation of other
lipases and esterases. In support of this model, analysis of systematic
truncations of PAF acetylhydrolase revealed that deletions beyond 54
amino acids from the NH
terminus and 21 from the COOH
terminus resulted in a loss of enzyme activity. These observations
demonstrate that although plasma PAF acetylhydrolase is a phospholipase
A
it has structural properties characteristic of the
neutral lipases and esterases.
Platelet activating factor (PAF) ()is a potent
pro-inflammatory phospholipid produced by activated platelets,
leukocytes, and endothelial cells(1) . Its production is
tightly regulated, both at the synthetic and degradative levels. PAF is
synthesized in activated cells primarily through the remodeling pathway
that invokes at least two enzymes, an arachidonatespecific
phospholipase A
(PLA
) and acetyl-CoA:lyso-PAF
acetyltransferase, both of which are activated by
phosphorylation(2, 3, 4, 5, 6) .
PAF is inactivated when the sn-2 ester bond is hydrolyzed by
PAF acetylhydrolase(s), of which there are both intracellular and
extracellular forms(7, 8, 9, 10) .
The cytoplasmic enzyme may help regulate PAF production (e.g. in differentiated macrophages(11) ), whereas the plasma
form is believed to regulate baseline circulating PAF levels and may be
critical in resolving inflammation(12) .
We recently cloned the plasma PAF acetylhydrolase cDNA(13) , and it encodes a 441-amino acid protein including a predicted signal peptide. Recombinant enzyme exhibited a marked preference for phospholipids with short chain moieties at the sn-2 position and blocked PAF-induced inflammation. The deduced amino acid sequence is unique except for the Gly-Xaa-Ser-Xaa-Gly (GXSXG) motif found in most serine esterases and lipases(14, 15, 16) . The central Ser of this motif serves as the active site nucleophile of these enzymes (reviewed in (17) ). The GXSXG motif in PAF acetylhydrolase is consistent with observations that the activity can be blocked by the active site Ser-specific inhibitor, diisopropyl fluorophosphate(7, 13) .
PAF acetylhydrolase is a
PLA because it hydrolyzes phospholipids at the sn-2 position. More than 60 secretory PLA
s and at
least two classes of intracellular PLA
s have been
described(18, 19) . The secretory PLA
s are
small (119-143 amino acids), have a high disulfide bond content
(10 or 14 cysteines), and require calcium for catalysis. PAF
acetylhydrolase is larger than these PLA
s, has fewer
cysteines (five), and functions in the absence of calcium. The
intracellular PLA
s are larger than the secreted ones and do
not utilize calcium as part of the catalytic
site(20, 21, 22, 23) . However, the
intracellular localization and the preference of these enzymes for
phospholipids with arachidonate or other long chain fatty acids at the sn-2 position distinguish them from the plasma PAF
acetylhydrolase. Thus, the unique functional properties of plasma PAF
acetylhydrolase likely reflect a molecular structure that is distinct
from that of other known PLA
s.
In contrast, the neutral
lipases share the calcium-independence of PAF acetylhydrolase and
contain the conserved GXSXG motif. The
three-dimensional structure of several of these lipases from species as
divergent as mammals, fungi, and bacteria have been defined and found
to have a core /
hydrolase fold structure that is
characteristic of a variety of esterases and
hydrolases(17, 24) . The
/
hydrolase
structure consists of alternating
-helices and
-strands; the
-strands are arranged in a parallel fashion and form the central
core of the molecule(17, 24) . The active site
contains a catalytic triad comprised of the nucleophilic Ser
(GXSXG), an acidic residue that is usually Asp (Glu
in Candida rugosa and Geotrichum candidum lipases(25, 26, 27, 28) ), and
an invariant His. In most lipases, the triad lies within a hydrophobic
pocket that is exposed upon contact with the substrate (interfacial
activation) which results in an enhanced reaction rate. Analytically,
the property of interfacial activation is manifested in surface
dilution kinetics, a property that PAF acetylhydrolase shares with
other PLA
s and the neutral
lipases(7, 18) .
Clearly, PAF acetylhydrolase
exhibits characteristics of both the PLAs and the neutral
lipases. However, the fact that PAF acetylhydrolase contains the
characteristic serine esterase GXSXG motif and is
inhibited by the active site serine modifier, diisopropyl
fluorophosphate, suggests that mechanistically, the enzyme is more
closely related to the neutral lipases and other esterases than it is
to the classical PLA
s. To better understand the structural
basis for the functional characteristics of PAF acetylhydrolase, we
used site-directed mutagenesis and deletion analyses to identify
potential catalytic residues and the minimal active portion of the
molecule.
Deletion constructs were generated using exonuclease III (Promega) to create a series of nested deletions from both the 5` and 3` ends. The parent constructs were engineered by PCR using primers with specific restriction sites compatible with the exonuclease III system. For 5` deletions, a sense primer was designed to allow cloning the product in-frame with the E. coli thioredoxin gene (31) under transcriptional control of the tac promoter (32) in pUC19. This primer (Start 3) contained XbaI, SphI, and XhoI restriction sites immediately upstream of the codon encoding Ile-42. This primer was paired with the Stop 3 antisense 3` primer to generate the parent construct. For 3` deletions, a 3` antisense primer (Stop 3) containing translation termination codons in all 3 reading frames preceded by XbaI, SacI, and EcoRV restriction sites was paired with Start 6 to generate a parent construct which was cloned into the same pUC19-trp vector used for the site-directed mutants.
Nucleotide sequence of the PCR primers follows (all sequences are written in the 5`-3` direction; parenthetical S and A indicate sense or antisense orientation): Start 1 (S), TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG; Start 3 (S), GCTCTAGAGCATGCACTTCGTATGATTGCCTCGAGATACAAGTACTGATGGCTGCTGCAAG; Start 6 (S), AGACTAGTAATTATGATACAAGTATTAATGGCTGCTGCAAG; Stop 2 (A), ATTGATATCCTAATTGTATTTCTCTATTCCTG; Stop 3 (A), CAGAGATATCAGTCAGTCAGAATAGAGCTCTGCAATCTTACGAAGTTCTAGAATTGTATTTCTCTATTCCTGAAGAGTTCTGTAACATGATG; Stop 4 (A), CGTGGTACCTCATTAATTGTATTTCTCTATTCCTG; S108A (S), TGGGGTCTTGCCAAATTTCTTGG; S108A (A), AGCCAGTGTGTTCCAAGAAATTTGGCAAGACC; S273A (S), TTGGACATGCTTTTGGTGGAG; S273A (A), TGCTCCACCAAAAGCATGTCCAATTACTGC; D286A (S), CAGACTCTTAGTGAAGCTCAGAGATTCAGATGTGG; D286A (A), CTGAATCTCTGAGCTTCACTAAGAGTCTG; D286N (S), CAGACTCTTAGTGAAAATCAGAGATTCAGATGTGG; D286N (A), CTGAATCTCTGATTTTCACTAAGAGTCTG; D296A (S), TATTGCCCTGGCTGCATGGATGTTTCCAC; D296A (A), CATGCAGCCAGGGCAATACCAC; D296N (S), TATTGCCCTGAATGCATGGATGTTTCCAC; D296N (A), CATGCATTCAGGGCAATACCAC; D304A (S), CACTGGGTGCTGAAGTATATTCCAGAATTCCTCAG; D304A (A), ATATACTTCAGCACCCAGTGGAAACATCCATGC; D338A (S), GCTACTCACCTGCTAAAGAAAGAAAGATG; D338A (A), CATCTTTCTTTCTTTAGCAGGTGAGTAG; H351A (S), GGGGTTCAGTCGCCCAGAATTTTGCTG; H351A (A), TTCTGGGCGACTGAACCCCTGATTGTAATC; H395A,H399A (S), GGCTTTAGGACTTGCTAAAGATTTTGATCAGTGGG; H395A,H399A (A), CTTTAGCAAGTCCTAAAGCCTTTTGTAAGAATGC. The sequence integrity of all constructs generated by PCR was confirmed by nucleotide sequencing.
Figure 1: Amino acid sequence alignment of PAF acetylhydrolase from multiple species. Sequences are deduced from cDNA clones obtained from phage cDNA libraries by low stringency hybridization to the human cDNA. The conserved GXSXG motif is underlined. Dots represent identity to the human sequence and dashes indicate spaces introduced to optimize the alignment. Residues identified by mutational analyses to be essential for human PAF acetylhydrolase catalytic activity are marked (*).
Figure 2: PAF acetylhydrolase specific activity in lysates of E. coli expressing mutated enzyme. Site-directed mutations were introduced into the human PAF acetylhydrolase cDNA by PCR. The mutated products were expressed in E. coli under control of the trp promoter and enzyme activity levels were measured in lysates of overnight cultures of transformants. A unit of activity is defined as 1 µmol of PAF hydrolyzed per ml/h and the quantity of each recombinant protein was determined by Western analysis as described under ``Experimental Procedures.'' These data are representative of three separate experiments. The relative positions of the mutations in the PAF acetylhydrolase protein are shown schematically below the graph. Residues essential for catalysis are indicated above the bar; all others are below.
Identifying the position of the active site Ser
provided a landmark from which the positions of the Asp and His
components of a catalytic triad might be predicted. We compared the
linear orientation of triad residues in lipases with defined structures (Table 3). Although these lipases are from phylogenetically
diverse organisms, they all have the basic /
hydrolase core
structure with the catalytic triad formed at one edge of the central
-sheet(24) . In each case, the linear order of the active
site residues is Ser, acidic residue, His. In these enzymes, the Ser
and the acidic residue are generally separated by either one or two
-strands and a variable number of
-helices, and the
complexity of this region correlates with the distance between the two
residues. Specifically, in both human pancreatic lipase and human
lipoprotein lipase, the catalytic Asp lies 24 residues downstream of
the Ser. The sequence between these residues contains an
-helix
followed by a
-strand(34, 35) . The structural
constraints required to maintain the
/
organization and to
position the Ser and Asp in close proximity may dictate a minimal
distance of approximately 24 amino acids between the two residues. In
most of the other lipases shown in Table 3, the greater distance
between the Ser and Asp correlates with the presence of two
-strands and a variable number of
-helices. These generalized
observations were used to predict the location of possible active site
Asp residues in plasma PAF acetylhydrolase. The structural
characteristics of the region between the active site Asp and His
residues of lipases are highly variable and consequently the spacing
between these residues has less predictive value.
The third parameter used to evaluate all potential Asp and His catalytic site residues was whether they were conserved in PAF acetylhydrolase from several species. Downstream from Ser-273 in the human sequence lie five His residues which could potentially be part of the catalytic triad. Three of these (His-367, His-399, and His-428) are not conserved in the other species (Fig. 1) and are therefore unlikely candidates. In contrast, there are nine conserved Asp residues carboxyl to Ser-273. We focused first on identifying the essential His then examined the Asp residues found between that His and Ser-273.
Both of the conserved
His residues downstream of Ser-273 were mutated to Ala. The H351A
mutation was expressed as an independent construct; however, because of
the close proximity of His-395 and His-399 and concerns of potential
substitution effects, mutations of both residues were expressed in a
single construct (H395A,H399A). As shown in Fig. 2, H351A lacked
any detectable PAF acetylhydrolase activity while H395A,H399A produced
more than wild-type levels of activity. As with the Ser mutations, both
His mutations reacted well with the monoclonal antibodies, suggesting
that these mutations did not grossly alter the conformation of the
enzyme (Table 2). The distance of 78 amino acids between His-351
and Ser-273 is less than that of any of the lipases in Table 3but is similar to the distance separating the active site
serine and histidine residues of a Pseudomonas dienlactone
hydrolase, a thiol esterase with an /
core and a catalytic
triad(36) . From these results, we conclude that His-351 is the
active site histidine in plasma PAF acetylhydrolase.
If PAF acetylhydrolase has the prototypical lipase structure, the active site Asp would be expected to lie between Ser-273 and His-351. Within this region are four conserved or semi-conserved Asp residues (Asp-286 is Glu in chicken; Asp-304 is Glu in mouse). Each was mutated to Ala and evaluated for an effect on PAF acetylhydrolase activity. The two mutations most distal to Ser-273, D304A and D338A, had no adverse effect on the activity of the enzyme (Fig. 2). In contrast, the D296A mutation reduced enzyme activity by more than 99% and D286A reduced the activity by more than 96% ( Fig. 2and Table 2). Since this loss of activity could arise from either an absolute requirement for both residues for catalysis or simply from a conformational perturbation introduced by one or both of the mutations, a more conservative mutation, Asp to Asn, was introduced at both positions. Again, mutation of Asp-296 (D296N) greatly reduced enzyme activity (by more than 99%). The D286N mutation, however, restored activity to within 70% of wild-type levels ( Fig. 2and Table 2). This suggests that the Asn mutations were less disruptive to conformation than the Ala mutations. This suggestion is supported by the observation that the Asp to Ala mutations rendered the proteins unrecognizable to the monoclonal antibodies but the Asp to Asn mutations restored the ability of the antibodies to bind the proteins (Table 2). Together, these data support the conclusion that Asp-296 is the acidic component of the catalytic triad. Interestingly, the distance of 23 amino acids between the active site Ser-273 and Asp-296 is similar to that of the pancreatic lipase family (24 amino acids).
Figure 3:
Amino-terminal heterogeneity of purified
plasma PAF acetylhydrolase. The enzyme was partially purified from
human plasma on two separate occasions as described previously (13) and the NH-terminal sequence was determined by
Edman degradation. The purification procedures were identical except
that fresh plasma was used in purification 2 whereas plasma used in
purification 1 was stored at 4 °C for up to 1
month.
Figure 4:
PAF
acetylhydrolase activity produced in E. coli by enzyme
truncated at the NH or COOH termini. A series of nested
truncations of the PAF acetylhydrolase cDNA was generated using an
exonuclease deletion system. NH
-terminal deletions were
expressed as fusions with the prokaryotic thioredoxin protein whereas
the COOH-terminal truncations were directly expressed. The
NH
- or COOH-terminal amino acid of each deletion is
indicated below the abscissa. The specific activity
values were determined as in Fig. 2and represent the results of
three independent experiments. The deletions are schematically
summarized below the graph. Deletions that do not negatively
affect enzyme activity are indicated above the bar, all others
are below. In addition, the initiating Met (M1) and the
NH
-terminal residue of the enzyme after cleavage of the
putative signal peptide (V18(13) ) are indicated. The shaded region of the bar represents the minimal
functional portion of the enzyme as defined by the
deletions.
Plasma PAF acetylhydrolase is a unique extracellular
PLA that abolishes the diverse, potent actions of PAF and
oxidized phospholipids. The enzyme is intriguing from a biochemical
viewpoint for several reasons. First, unlike the other secreted
PLA
s, PAF acetylhydrolase does not require calcium for
activity(7) . Another reason is the marked specificity the
enzyme has for short chain sn-2 moieties (37) . This
specificity has the important advantage of allowing the enzyme to
circulate in a completely active form, unlike the plasma proteases,
which circulate as inactive zymogens. The basis for the specificity is
unknown, but suggests that PAF acetylhydrolase has a substrate-binding
region that excludes longer acyl chains. The structures of several
extracellular PLA
s have been solved at high resolution and
they have been shown to have a hydrophobic pocket into which the acyl
chains of the substrate phospholipid fit, positioning the sn-2
ester at the active site(19, 38) . The structure of
PAF acetylhydrolase is expected to be quite distinct because of its
strict specificity for the short sn-2 residues. The
observations reported here are consistent with this notion and
illuminate another intriguing feature of the enzyme: mechanistically,
plasma PAF acetylhydrolase appears to utilize a Ser-Asp-His catalytic
triad and may assume the classical
/
hydrolase conformation
of the neutral lipases. This enzyme is the first PLA
purported to exhibit these characteristics. The catalytic site of
the secretory PLA
s contains an essential Asp and a His but
no Ser. Rather, a calcium ion is used to stabilize the transition
state(18) . In contrast, a cloned intracellular PLA
does not require calcium for catalysis but neither does it have
the conserved pentapeptide, GXSXG, that is
characteristic of the neutral
lipases(39, 40, 41) . Thus, plasma PAF
acetylhydrolase is functionally a PLA
but is structurally
and mechanistically distinct from the other known PLA
s. It
is possible that another PAF-specific PLA
, the distinct,
cytoplasmically active PAF acetylhydrolase described by Hattori et
al.(9, 10) , has similar structural features
since it also contains a (semi)conserved GXSXG motif.
The evidence for the presence of a catalytic triad in PAF
acetylhydrolase is compelling. Previous experiments have demonstrated
that activity of the enzyme is inhibited by diisopropyl
fluorophosphate, an agent that modifies active site Ser
residues(7) . Cloning of the plasma PAF acetylhydrolase gene
revealed that the enzyme has the characteristic lipase/serine protease
consensus pentapeptide, GXSXG(13) , which
generally contains the active site Ser. In the current report we have
demonstrated by site-directed mutagenesis that the Ser within this
motif is required for activity. The presence of a catalytically
essential His was first suggested by experiments in which a modifier of
active site His residues, p-bromophenacylbromide, was used to
inhibit the activity of PAF acetylhydrolase(42) . Based upon
the linear configuration of catalytic residues in other lipases, the
His as well as the acidic components of a triad would be expected to
lie carboxyl to Ser-273. There are only two conserved His residues
within this region; mutation of one, His-351, destroyed enzyme activity
while mutation of the other did not affect function of the enzyme.
Identification of the acidic triad component was complicated by the
fact that between Ser-273 and His-351 lie four conserved Asp residues,
but multiple analyses revealed that only Asp-296 was essential for
hydrolytic activity. The distance of 23 amino acids between the
essential Ser-273 and Asp-296 is interesting because it is very similar
to the spacing (24 amino acids) between the analogous residues in the
pancreatic lipase family (Table 3). Within this region in
pancreatic lipase as well as in other lipases, the
GXSXG motif forms a tight ``nucleophilic
elbow'' at the immediate carboxyl end of a -strand (24) . This is followed by one
-helix, another
-strand, and then the catalytic Asp. This distinguishes the
secondary structure of the pancreatic lipase family from that of other
lipases or hydrolases in which at least two
-helix/
-strand
pairs separate the Ser and acidic residues. Therefore, by
extrapolation, we predict that the secondary structure of this region
in plasma PAF acetylhydrolase may resemble that of the pancreatic
lipase family.
The evidence for an /
hydrolase
conformation of plasma PAF acetylhydrolase is inferential. The enzyme
lacks any sequence homology with other lipases except for the
GXSXG motif. In the lipases, the Ser within this
motif functions as the essential nucleophilic component of the
catalytic triad. The presence of the conserved pentapeptide and other
components of an apparent catalytic triad as well as the lipid
specificity of PAF acetylhydrolase place it on common ground with other
lipases. In addition, the intriguing linear similarity between PAF
acetylhydrolase and the pancreatic lipase family within the active site
Ser-Asp region is consistent with the enzyme assuming a similar
secondary structure. Taken together, our results are consistent with
the hypothesis that the enzyme is an
/
hydrolase with a
catalytic triad, but confirmation of this hypothesis awaits
crystallization and x-ray structural analysis.
We have demonstrated
that plasma PAF acetylhydrolase is a PLA with mechanistic
and structural characteristics of the neutral lipases. Also like these
lipases, PAF acetylhydrolase displays the property of interfacial
activation (7) which, in the lipases, has been tied to the
presence of a ``lid'' comprised of
-helices or loops
that cover the hydrophobic catalytic pocket when the enzyme is in the
inactive state(43, 44, 45) . Upon contact
with a lipid surface, conformational rearrangements occur that expose
the active site to the substrate. Enzymes lacking the lid over the
catalytic site are not activated at the lipid-water interface (guinea
pig pancreatic phospholipase (46) and fungal
cutinase(47) ). Given the other similarities between PAF
acetylhydrolase and the lipases, the enzyme might also be expected to
have a lid-like structure that mediates its interfacial properties.
The nucleotide
sequences of the cDNAs encoding the protein sequences reported in this
paper have been submitted to the GenBank/EMBL
Data Bank with accession numbers U34247 [GenBank](bovine), U34246 [GenBank](dog), U34277 [GenBank](mouse), and U34278 [GenBank](chicken).