Molecular Basis of the Interaction between Plasma
Platelet-activating Factor Acetylhydrolase and Low Density
Lipoprotein*
Diana M.
Stafforini
§,
Larry W.
Tjoelker¶,
Sally P. A.
McCormick
,
Darius
Vaitkus
,
Thomas M.
McIntyre**,
Patrick W.
Gray¶,
Stephen G.
Young
, and
Stephen M.
Prescott
From the
Huntsman Cancer Institute, Program in Human
Molecular Biology and Genetics, Eccles Institute of Human Genetics, and
** Cardiovascular Research and Training Institute, University of Utah,
Salt Lake City, Utah 84112, ¶ ICOS Corp., Bothell, Washington
98021, and
Gladstone Institute of Cardiovascular Disease,
Cardiovascular Research Institute, and Department of Medicine,
University of California, San Francisco, California 94141-9100
 |
ABSTRACT |
The platelet-activating factor acetylhydrolases
are enzymes that were initially characterized by their ability to
hydrolyze platelet-activating factor (PAF). In human plasma, PAF
acetylhydrolase (EC 3.1.1.47) circulates in a complex with low density
lipoproteins (LDL) and high density lipoproteins (HDL). This
association defines the physical state of PAF acetylhydrolase, confers
a long half-life, and is a major determinant of its catalytic
efficiency in vivo. The lipoprotein-associated enzyme
accounts for all of the PAF hydrolysis in plasma but only two-thirds of
the protein mass. To characterize the enzyme-lipoprotein interaction,
we employed site-directed mutagenesis techniques. Two domains within
the primary sequence of human PAF acetylhydrolase, tyrosine 205 and
residues 115 and 116, were important for its binding to LDL. Mutation
or deletion of those sequences prevented the association of the enzyme with lipoproteins. When residues 115 and 116 from human PAF
acetylhydrolase were introduced into mouse PAF
acetylhydrolase (which normally does not associate with LDL), the
mutant mouse PAF acetylhydrolase associated with lipoproteins. To
analyze the role of apolipoprotein (apo) B100 in the formation of the
PAF acetylhydrolase-LDL complex, we tested the ability of PAF
acetylhydrolase to bind to lipoproteins containing truncated forms of
apoB. These studies indicated that the carboxyl terminus of apoB plays
a key role in the association of PAF acetylhydrolase with LDL. These
data on the molecular basis of the PAF acetylhydrolase-LDL association
provide a new level of understanding regarding the pathway for the
catabolism of PAF in human blood.
 |
INTRODUCTION |
Platelet-activating factor
(PAF)1 is a phospholipid
messenger synthesized by a variety of cells involved in host defense,
such as endothelial cells, neutrophils, and monocytes (1). PAF
functions both in normal physiological events and in pathological
responses, particularly inflammation and allergy (1). High levels of
PAF are associated with a variety of human diseases such as asthma, necrotizing enterocholitis, and sepsis, as judged by direct measurement of PAF levels (2-5), by the effects of PAF receptor antagonists (6-9), and by the effects of an enzyme that inactivates PAF (10, 11).
PAF is inactivated by hydrolysis of the sn-2 acetyl group, a
reaction catalyzed by PAF acetylhydrolases (12, 13). The secreted form
of PAF acetylhydrolase circulates in human plasma as a hydrophobic
protein complexed with low density lipoproteins (LDL) and high density
lipoproteins (HDL) (14, 15).
In addition to defining the physical state of PAF acetylhydrolase in
the plasma compartment, the association of this enzyme with
lipoproteins has important implications for catalysis. The association
of the enzyme with LDL is a major determinant of its catalytic
efficiency in vivo; when the substrate
concentration is limiting, the LDL-associated activity accounts for
virtually all of the PAF hydrolysis in plasma (16). Thus, the failure of PAF acetylhydrolase to bind to LDL would be predicted to block the
physiological function of the enzyme, even if its intrinsic catalytic
activity remained intact. In addition, the association of PAF
acetylhydrolase with lipoproteins allows this relatively hydrophobic
enzyme to circulate in blood and gain unfettered access to sites of
inflammation and cellular activation. Finally, it is possible that the
lipoprotein lipids might facilitate access of hydrophobic lipid
substrates to the active site of the enzyme, which may account for the
efficiency of substrate hydrolysis when the enzyme is bound to
lipoproteins. Therefore, identifying the factors that define PAF
acetylhydrolase's association with lipoproteins is an important issue
with clear cut physiologic consequences.
In this study, we characterized the molecular basis for the interaction
between PAF acetylhydrolase and LDL. Using site-directed mutagenesis,
we identified two domains of PAF acetylhydrolase that are important for
its interaction with LDL. In addition, we tested the hypothesis that
the principal protein component of LDL, apolipoprotein (apo) B100, is
important for the PAF acetylhydrolase-LDL interaction by analyzing the
binding of PAF acetylhydrolase to lipoproteins containing truncated
forms of human apoB. Our data indicate that the carboxyl terminus of
human apoB100 plays a key role in the association of PAF
acetylhydrolase with LDL.
 |
EXPERIMENTAL PROCEDURES |
Materials--
PAF(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
was purchased from Avanti Polar Lipids (Birmingham, AL). A
polyclonal antibody against PAF acetylhydrolase was prepared by
immunizing New Zealand White rabbits with the purified recombinant
protein (Quality Controlled Biochemicals, Hopkinton, MA).
[acetyl-3H]PAF was from NEN Life Science Products.
Site-directed Mutagenesis and
Deletions--
Oligonucleotide-directed mutagenesis of various sites
within PAF acetylhydrolase was performed as described (11). The
presence of the desired mutations or deletions was confirmed by
automated analysis of DNA sequences. All mutants were expressed in a
pUC vector to which the tryptophan promoter was added to allow
expression of PAF acetylhydrolase in Escherichia coli (11).
The promoter was derepressed by depletion of tryptophan during
overnight incubation at 37 °C. The cultures were harvested by
centrifugation, and the cells were resuspended in buffer A (100 mM succinate, 100 mM NaCl, 1 mM
EDTA, 20 mM CHAPS, pH 6.0), lysed by sonication, and
allowed to incubate on ice for 60 min. The soluble fraction was
recovered by centrifugation, and the pellet was discarded. Supernatant
fluids were assayed for PAF acetylhydrolase activity as described below and subjected to immunoblot analysis (11). Recombinant PAF
acetylhydrolase was detected with a polyclonal anti-PAF
acetylhydrolase antiserum (diluted 1:2000) as the primary antibody and
a horseradish peroxidase-labeled goat anti-rabbit IgG antibody
(diluted 1:5000) from Kirkegaard and Perry Laboratories (Gaithersburg,
MD). The blots were developed with the enhanced chemiluminescence (ECL)
system (Amersham Pharmacia Biotech).
Assessment of PAF Acetylhydrolase Binding to LDL--
Human LDL
were isolated from the plasma of healthy subjects by a single spin
ultracentrifugation technique (15). The endogenous PAF acetylhydrolase
activity in the LDL fraction was irreversibly inactivated by treatment
with 5.0 mM diisopropylfluorophosphate (DFP, Sigma) for 60 min at 37 °C and dialysis against phosphate-buffered saline. The
ability of mutant and wild-type PAF acetylhydrolase proteins to
associate with LDL was assessed by incubating DFP-treated LDL (DFP-LDL)
with 5 µg of solubilized extract from E. coli for 2 h
at 37 °C. In control experiments, the amount of CHAPS carried over
to the assay had no effect on the association of endogenous PAF
acetylhydrolase activity with LDL or the ability of exogenously added
PAF acetylhydrolase to bind to DFP-LDL. After the incubation, the
mixtures of DFP-LDL and E. coli extracts were subjected to ultracentrifugation, as above. The LDL fraction and the
lipoprotein-free "bottom fraction" were collected and assayed for
PAF acetylhydrolase activity by incubation with
[acetyl-3H]PAF and separation of the reaction
products by reverse phase liquid chromatography, as described by
Stafforini et al. (17). A unit was defined as a µmol of
substrate hydrolyzed per h at 37 °C. LDL binding was assessed by
calculating the fractional area in the LDL peak with Matlab software.
The generation and characterization of transgenic mice expressing the
full-length human apoB100 have been reported (18). Transgenic mice
expressing truncated versions of apoB (apoB80 (apo B amino acids
1-3619; Ref. 19), and apoB90 (apoB amino acids 1-4084; ref. 20)) have
also been described (the expression of mutant human apoB proteins in
transgenic mice is reviewed in Ref. 21). Plasma from these mice was
isolated and stored at 4 °C. Endogenous PAF acetylhydrolase in the
plasma was inactivated with DFP (5.0 mM, 60 min at
37 °C). The samples then were dialyzed on Centricon 50 microconcentrators (Amicon, Inc., Beverly, MA) and incubated with
recombinant PAF acetylhydrolase or with 5 µg of a solubilized extract
of E. coli that had been transformed with the wild-type PAF
acetylhydrolase cDNA.
 |
RESULTS |
The Amino and Carboxyl Termini of PAF Acetylhydrolase Are Not
Necessary for Association with LDL--
Our initial studies were aimed
at establishing the minimal segment of the PAF acetylhydrolase molecule
required for specific binding to LDL. We tested several deletion
constructs for their ability to associate with LDL in human plasma or
with purified LDL fractions that had been pretreated with DFP to
inhibit endogenous PAF acetylhydrolase activity. Deletion of up to 60 amino acids from the amino terminus and 21 amino acids from the
carboxyl terminus of PAF acetylhydrolase had no effect on the ability
of these constructs to bind to LDL (>80% of the total activity was
associated with LDL in both cases). Further deletions abolished
enzymatic activity (22). Therefore, we did not use the deletion
approach to test other domains in the PAF acetylhydrolase molecule.
Tyrosine 205 Is Required for Binding of PAF Acetylhydrolase to
LDL--
Next, we systematically mutated all tyrosine residues in the
PAF acetylhydrolase molecule to phenylalanines. Tyrosines were analyzed
because they are important in the interaction of proteins with
macromolecules in other systems (23). Tyrosine 440 was not analyzed
because it lies within the carboxyl-terminal 20 amino acids of the
protein, which has no effect on LDL binding. Mutation of tyrosine
residues 20, 26, 63, 84, 85, 103, 144, 160, 188, 189, 307, 321, 324, and 335 had no effect on enzymatic activity or immunoreactivity (data
not shown). All but one of the tyrosine mutants bound to purified,
DFP-treated human LDL in a manner similar to the wild-type protein
(>75%). Mutant Y205F had decreased binding (59%, Fig.
1), suggesting that residue 205 might be
important for the interaction. A mutant with alanine at this position
(Y205A) did not bind to LDL (Fig. 1). To determine if other residues in the region are also important for the interaction, we generated alanine
mutants of the six adjacent amino acid residues. Changing residues
202-204 and 206-208 to alanines had no effect on enzymatic activity
or on the association with LDL (not shown). Therefore, a conformational
change resulting from the replacement of tyrosine at position 205 with
alanine is unlikely to account for altered binding to LDL.

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Fig. 1.
Tyrosine 205 is involved in the interaction
between PAF acetylhydrolase (AH) and LDL. Three
PAF acetylhydrolase constructs (wild-type (WT) PAF
acetylhydrolase (A); Y205F (B); and Y205A
(C)) starting from the initiating methionine were cloned
into a pUC 19 vector under the control of a tryptophan promoter and
expressed in E. coli, as described (22). After harvest, the
cells were resuspended in CHAPS-containing solubilization buffer, and
the insoluble material was removed by centrifugation. The extracts were
incubated with DFP-inactivated human LDL particles and subjected to
density gradient ultracentrifugation. Fractions were collected and
assayed for PAF acetylhydrolase activity, as described by Stafforini
et al. (17). The results are representative of two
experiments performed under identical conditions.
|
|
Tyrosine 205 is present in all species examined to date, including
human, bovine, dog, chicken, and mouse (22). When we tested the ability
of the dog and mouse recombinant PAF acetylhydrolases to bind human
LDL, the dog protein associated with LDL in a normal fashion, but the
mouse protein did not (Fig. 2). These
observations suggest that tyrosine 205 is necessary but not sufficient
for binding to LDL and that an additional region or regions are
involved in the interaction of the enzyme with LDL.

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Fig. 2.
Association of dog and mouse PAF
acetylhydrolase (AH) with human LDL. The
cDNAs encoding dog (A) and mouse (B) PAF
acetylhydrolase homologs (22) were cloned into the pUC 19 and expressed
from the initiating methionine, as described in the legend to Fig. 1.
Binding to human LDL was assessed after incubation with the
lipoprotein, ultracentrifugation, fractionation, and enzymatic activity
determinations, as described under "Experimental Procedures." The
results are representative of two experiments performed under identical
conditions.
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|
An Amino-terminal Region in PAF Acetylhydrolase Is Necessary for
Binding to LDL--
Next, we tested the possibility that histidine
residues in PAF acetylhydrolase are important for binding to LDL. We
previously observed that the association of this enzyme with LDL is
dependent on pH and that its pH profile is consistent with the notion
that a histidine residue plays a role in the process (15). All
histidines (residues 7, 32, 74, 114, 152, 170, 179, 216, 241, 272, 367,
395, 399, and 428) were changed to alanines (except histidine 351, which is part of the catalytic triad in PAF acetylhydrolase (22)), and
the resulting mutants were tested for binding to LDL. In general, mutation of histidines to alanines had no effect on the enzymatic activity of the recombinant products. However, changing histidine 272 to alanine abolished enzymatic activity. Histidine 272 is next to
serine 273, which is part of a catalytic triad that is characteristic
of lipases (22). It is not surprising that the H272A mutant expressed
no enzymatic activity, because altering the conformation of the protein
in this region would be likely to disrupt the three-dimensional
structure near the active site. Western blot analysis of the histidine
mutants showed expression of protein at levels corresponding to the
activity in the samples (not shown).
In LDL binding assays, all of the histidine mutants had normal binding
except for H114A, which had altered binding to LDL (Fig.
3). Replacement of histidine 114 with
glutamate or glutamine also abolished binding to LDL (Fig. 3),
suggesting that the effect was independent of charge. Further analysis
indicated that a residue or residues important for binding to LDL exist
near histidine 114, and several findings suggest that histidine 114 is
not directly involved. First, a deletion mutant lacking histidine 114 was enzymatically active and bound normally to LDL (Fig. 3). Second,
the dog homolog, which has a proline at position 114, also bound
normally to LDL (Fig. 2). Third, the mouse PAF acetylhydrolase protein,
which has a proline residue at position 113 (equivalent to histidine 114 in the human molecule), did not associate with human LDL (Fig. 2B); however, mutation of proline 113 in the mouse sequence
to histidine did not result in binding (Fig. 3). Thus, while histidine 114 in human PAF acetylhydrolase is near a region that is important for
binding, it is not required for the association with LDL.

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Fig. 3.
Effect of histidine 114 on binding of PAF
acetylhydrolase (AH) to LDL. The role of
histidine 114 was examined by comparing the binding of wild-type PAF
acetylhydrolase to LDL with that of three mutants in which the residue
was mutated (H114A, H114Q, and H114E) or deleted (H114ø). The mouse
PAF acetylhydrolase homolog was used to generate a mutant in which the
proline present at position 113 (equivalent to position 114 in the
human cDNA) was replaced with a histidine residue. The results are
representative of two experiments performed under identical
conditions.
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To identify the residue or residues near histidine 114 that are key for
binding of PAF acetylhydrolase to LDL, we mutated the neighboring
residues to alanines and expressed the mutant constructs in E. coli. The recombinant proteins were expressed at levels comparable
with those of the wild-type construct (Fig. 4A). Mutation of residues on
the amino-terminal side of histidine 114 had little effect on binding
(Fig. 4). In contrast, replacement of tryptophan 115, leucine 116, and
methionine 117 with alanine altered the association with LDL (Fig.
4B). Mutation of tryptophan 115 had the greatest effect, but
changing leucine 116 and methionine 117 also altered binding. We next
generated additional constructs in which tryptophan 115, leucine 116, and methionine 117 were individually deleted (Fig. 4C). We
found that these deletion mutants retained expression of enzymatic
activity, but binding to LDL was severely reduced (Fig. 4C).
Thus, in contrast to histidine 114 (Fig. 3), the deletion of tryptophan
115, leucine 116, and, to a lesser extent, methionine 117 resulted in
molecules that lacked the ability to associate with LDL, indicating
that these residues are necessary for binding to the lipoprotein.

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Fig. 4.
Mutation and deletion of residues in the
carboxyl-terminal end of histidine 114 affects binding of PAF
acetylhydrolase (AH) to LDL. Seven residues that
border histidine 114 on the amino- and carboxyl-terminal ends were
individually changed to alanines, and the resulting mutants as well as
the wild-type (WT) constructs were expressed in E. coli. After solubilization (described in the legend to Fig. 1), we
determined enzymatic activities and protein contents (A). We
also analyzed the ability of each construct to bind to LDL
(B and C). The results are representative of two
experiments performed under identical conditions.
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The role played by tryptophan 115 and leucine 116 in the binding of PAF
acetylhydrolase to LDL was confirmed in the following experiment. We
made use of the fact that the primary sequence of the mouse PAF
acetylhydrolase in the region of residues 114-117 is quite different
from the human sequence (Fig. 5). We
systematically mutated the residues in the mouse PAF acetylhydrolase to
mimic the domain present in the human molecule. As the homology in this region was increased to match that of the human PAF acetylhydrolase, binding to LDL was restored (Fig. 5). Changing proline 113 to histidine
did not significantly affect binding (1% for the wild-type versus 7.5% for the P113H mutant). Replacing proline 113 and serine 114 to histidine and tryptophan, respectively, increased
binding to LDL to 40.9%. When proline 113, serine 114, and isoleucine 115 of the mouse enzyme were converted to histidine, tryptophan, and
leucine, respectively, binding to LDL increased to 64.4%. Notably, the
dog PAF acetylhydrolase (which binds to human LDL) is identical to
human PAF acetylhydrolase in this region. Thus, site-directed
mutagenesis and deletion analyses showed that residues 115 and 116 are
critical for binding of PAF acetylhydrolase to LDL. Moreover, the
introduction of these residues into the mouse sequence yielded an
enzyme that was capable of binding to LDL (unlike the wild-type mouse
enzyme). Methionine 117 was not essential for binding to LDL (Fig.
5).

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Fig. 5.
Matching the mouse PAF acetylhydrolase
(AH) residues near proline 113 to the human sequence
near histidine 114 results in binding to LDL. A region near
proline 113 in the mouse PAF acetylhydrolase sequence was progressively
changed to generate mutants with sequences that partially (or
completely) matched that of the human enzyme in this region. The
results are representative of two experiments performed under identical
conditions.
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Cysteine Residues Are Not Involved in the PAF Acetylhydrolase-LDL
Interaction--
The full-length PAF acetylhydrolase cDNA encodes
eight cysteines (22), but the mature form of the enzyme is predicted to contain only five cysteines (22). Therefore, the mature protein is
likely to contain at least one unpaired sulfhydryl group, which might
be involved in binding to LDL. In addition, PAF acetylhydrolase has
been reported to have lower affinity for LDL than for lipoprotein(a) (24), which differs from LDL by the covalent attachment of apo(a), a
glycoprotein that is highly homologous to plasminogen (25). Apo(a)
associates with apoB by a disulfide bond (26, 27). To examine the
possibility that PAF acetylhydrolase and LDL interact through a
disulfide bond, we tested the effect of reducing agents on the
distribution of PAF acetylhydrolase in lipoproteins. Relatively high
concentrations of dithiothreitol (1, 5, and 10 mM) had no effect on enzymatic activity or association with LDL (not shown). The
failure of dithiothreitol to dissociate PAF acetylhydrolase and LDL
makes it very unlikely that their interaction depends on a disulfide
bond. However, to further explore this issue, we generated five mutants
in which the cysteine residues were converted to serines. Cysteine
residues 11, 13, and 15 were not examined, because they lie within a
region of the amino terminus shown by the deletion studies to be
unimportant for binding to LDL. We tested the ability of C67S, C229S,
C291S, C334S, and C407S to bind LDL. The mutants retained enzymatic
activity and associated with LDL in a manner identical to that of the
wild-type construct (data not shown).
The Carboxyl Terminus of ApoB100 Participates in the Association
between PAF Acetylhydrolase and LDL--
There is some evidence that
apoB100 is the protein component that mediates the binding of PAF
acetylhydrolase to LDL. First, our early studies on the purification of
PAF acetylhydrolase showed that a carboxyl-terminal fragment of apoB100
(including residues 4119-4536) co-purified with PAF acetylhydrolase
through numerous purification steps, strongly suggesting that the
carboxyl-terminal portion of apoB100 and PAF acetylhydrolase interact
with each other (28). Second, apoB100 is the major protein component of LDL. Third, in human subjects who are deficient in apoB100, PAF acetylhydrolase associates entirely with HDL (16). To determine which
domains in apoB100 are involved in the interaction of LDL with PAF
acetylhydrolase, we analyzed plasma from transgenic mice expressing
human apoB100, apoB90, or apoB80. The endogenous mouse PAF
acetylhydrolase associated exclusively with HDL in wild-type mouse
plasma. Wild-type mouse plasma contains very low levels of LDL (29).
However, the "HDL distribution" of mouse PAF acetylhydrolase was
also observed with plasma samples from all of the human apoB transgenic
mice (which have high levels of LDL), indicating that the endogenous
PAF acetylhydrolase does not bind to LDL particles containing either
full-length human apoB100 or the truncated human apoB proteins. This
result is consistent with our in vitro studies on the
association of mouse PAF acetylhydrolase with human LDL (Fig. 2).
To evaluate binding of human PAF acetylhydrolase to LDL produced by
transgenic mice, we inactivated the endogenous, HDL-associated PAF
acetylhydrolase activity in the plasma samples by pretreatment with
DFP. The pretreated plasma samples were then combined with purified
recombinant human PAF acetylhydrolase or with solubilized extracts from
bacteria overexpressing the human enzyme. After a 2-h incubation at
37 °C, the lipoproteins were fractionated by ultracentrifugation,
and each fraction was assayed for enzymatic activity. Human plasma
pretreated with DFP served as the control source of acceptor lipid
particles. When plasma derived from transgenic mice overexpressing
human apoB100 was used as the acceptor, PAF acetylhydrolase associated
with LDL and HDL (Fig. 6B) in
a manner virtually identical to that observed with human plasma (Fig.
6A). In contrast, when samples from control mice (not shown)
or mice expressing human apoB90 or human apoB80 were used as the source of lipoprotein acceptor particles, PAF acetylhydrolase associated exclusively with HDL (Fig. 6, C and D,
respectively). Identical results were obtained when the plasma
lipoproteins were separated by chromatography on Superose 6 columns
(not shown). These results indicate that the carboxyl-terminal portion
of apoB100 is required for binding of PAF acetylhydrolase to LDL.

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Fig. 6.
Binding of human PAF acetylhydrolase
(AH) to lipoproteins in plasma from transgenic mice
overexpressing various forms of human apoB100. Recombinant PAF
acetylhydrolase was incubated with DFP-inactivated human plasma and
subjected to ultracentrifugation and fractionation. The fractions then
were assayed for PAF acetylhydrolase activity (A).
Recombinant PAF acetylhydrolase was incubated with DFP-inactivated
plasma from mice overexpressing human apoB100 (B), apoB90
(C), or apo B80 (D). The lipoproteins were
separated by ultracentrifugation and then assayed for PAF
acetylhydrolase activity. The results are representative of five
experiments performed under similar conditions.
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 |
DISCUSSION |
This study demonstrates that the interaction between human PAF
acetylhydrolase and human LDL is likely to involve protein-protein interactions between specific residues in the plasma PAF
acetylhydrolase molecule and residues in apoB100, the principal
apolipoprotein of LDL. Mutation of tyrosine 205, which is completely
conserved among species (22), abolished binding to LDL. None of the
other tyrosine residues seem to be required for binding, because
replacing them with alanines did not affect the ability of the
recombinant proteins to associate with LDL. The fact that mutating
residues near tyrosine 205 did not affect binding to LDL suggests that the effect observed is specific to tyrosine 205 and not simply a
consequence of conformational changes in that region of the PAF
acetylhydrolase molecule. It is also possible that tyrosine 205 interacts with a distal site within PAF acetylhydrolase, inducing or
stabilizing a conformation necessary for binding to LDL. The inability
of mouse PAF acetylhydrolase to bind human LDL despite the presence of
tyrosine 205 is supportive of this possibility.
Several findings suggest that a region centered on tryptophan 115 and
leucine 116 is also involved in the interaction between PAF
acetylhydrolase and LDL. First, replacement of these residues with
alanines abolished binding to LDL. Second, deletion of these residues
also prevented association with the lipoprotein. Third, introduction of
these residues into the wild-type mouse PAF acetylhydrolase (which
normally does not associate with LDL) resulted in a mutant mouse enzyme
that bound to LDL almost as efficiently as the wild-type human enzyme.
The observation that tyrosine 205 as well as tryptophan 115 and
leucine 116 are necessary for binding to LDL suggests that these two
domains might have adjacent locations within the three-dimensional structure of the plasma form of PAF acetylhydrolase. However, an x-ray
crystallographic analysis of secreted PAF acetylhydrolase is not yet
available, and there is no homology of these domains to other PAF
acetylhydrolases or lipases whose three-dimensional structure is known
(30, 31).
Our findings also suggest that the carboxyl terminus of human
apoB100 is involved in binding to PAF acetylhydrolase. ApoB90 and
apoB80 lacked the ability to bind to PAF acetylhydrolase, consistent
with participation of the carboxyl terminus of apoB100 in the
interaction with the enzyme. Our results, however, do not completely
exclude the possibility that the structural features for the
interaction with PAF acetylhydrolase are present within apoB80 and
apoB90 but are not in the proper conformation for the interaction. For
example, altered composition of apoB90-containing lipoproteins might
change the conformation on the surface of the lipoprotein, affecting
its ability to interact with PAF acetylhydrolase. However, additional
evidence suggests that the carboxyl terminus of apoB100 and PAF
acetylhydrolase interact directly (i.e. without the
involvement of lipid components). In our early studies on the
purification of PAF acetylhydrolase, a carboxyl-terminal fragment of
apoB100 (including apoB100 residues 4119-4536) co-purified with PAF
acetylhydrolase, suggesting that a tight interaction exists between the
enzyme and this domain of apoB100 (28). Also, we have very recently
analyzed the ability of PAF acetylhydrolase to bind to mutant human LDL
particles (isolated from the plasma of transgenic mice) in which amino
acids 4279-4536 of the human apoB100 sequence were replaced by the
corresponding sequences from mouse apoB100. Interestingly, the mutant
LDL containing the human-mouse hybrid apoB bound to PAF
acetylhydrolase normally, suggesting that apoB100 residues 4279-4536
may not be critical for the interaction between PAF acetylhydrolase and
human apoB100.2 These
results, taken together with our earlier studies (28) and the current
studies on human apoB90 and human apoB80, suggest the possibility that
human apoB100 amino acids 4119-4279 could be important for binding to
PAF acetylhydrolase. That possibility will be tested in future studies.
It is interesting that mice, which have very low plasma concentrations
of LDL, also synthesize a PAF acetylhydrolase that is intrinsically
defective in its ability to bind to LDL. It is tempting to speculate
that these two features (low LDL levels and a "nonbinding" PAF
acetylhydrolase) may have arisen together during mammalian evolution;
it seems possible that the extremely low levels of LDL in mouse plasma
may have obviated any selective pressure to have a PAF acetylhydrolase
that binds to apoB100. In any case, the nonbinding mouse PAF
acetylhydrolase has provided us with a useful experimental tool, the
ability to analyze the binding of human PAF acetylhydrolase to human
apoB proteins without having to worry about interference from the
endogenous mouse protein. In humans, LDL-associated PAF acetylhydrolase
plays a key role in the clearance of PAF and PAF analogs from the
plasma, while the HDL-associated enzyme is less important (15). In
mice, the absence of LDL-associated PAF acetylhydrolase activity
appears to be compensated for by the HDL-associated enzyme; this could explain why the total serum PAF acetylhydrolase activity is
approximately 5 times higher in mice than in humans (32).
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ACKNOWLEDGEMENTS |
We are indebted to Dr. Guy A. Zimmerman for
insightful comments and critical review of this manuscript. We thank
Drs. T. L. Innerarity and J. Borén for providing human
apoB80 transgenic mice. We are grateful to Dr. Massimiliano Zaniboni
for performing area calculations, Diana Lim for expert assistance on
figure preparation, Stephen Ordway for making editorial comments on the
manuscript, and Connie Zlot for technical assistance. We also thank the
DNA Sequencing and Peptide/Oligo DNA Synthesis Core Facilities at the
University of Utah, which are supported by National Institutes of
Health Grant CA42014.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL35828 and HL41633 and by the Huntsman Cancer Institute.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
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§
To whom correspondence should be addressed: Program in Human
Molecular Biology & Genetics, 15 N. 2030 East, Rm. 2100, University of
Utah, Salt Lake City, UT 84112-5330. Tel.: 801-585-3402; Fax: 801-585-6345; E-mail: diana.stafforini{at}hci.utah.edu.
2
D. Stafforini, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
PAF, platelet-activating factor;
LDL, low density lipoprotein(s);
HDL, high
density lipoprotein(s);
DFP, diisopropylfluorophosphate;
apo, apolipoprotein;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
 |
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