Topology of the Stable Serpin-Protease Complexes Revealed by an
Autoantibody That Fails to React with the Monomeric Conformers of
Antithrombin*
Véronique
Picard
,
Pierre-Emmanuel
Marque
,
Francis
Paolucci§,
Martine
Aiach
, and
Bernard F.
Le
Bonniec
¶
From
INSERM, Unité 428, Université Paris
V, 75270 Paris Cedex 06 and § Sanofi Research,
34000 Montpellier, France
 |
ABSTRACT |
Solving the structure of the stable complex
between a serine protease inhibitor (serpin) and its target has been a
long standing goal. We describe herein the characterization of a
monoclonal antibody that selectively recognizes antithrombin in complex
with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's
reactive center loop (RCL, ultimately cleaved between the
P1 and P'1 residues). Accordingly, this
antibody reacts with none of the monomeric conformers of antithrombin
(native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge
stabilizing system has been swapped for alanine). The neoepitope
encompasses the motif DAFHK, located in native antithrombin on strand 4 of
-sheet A, which becomes strand 5 of
-sheet A in the
RCL-cleaved and latent conformers. The inferences on the structure of
the antithrombin-protease stable complex are that either a major
remodeling of antithrombin accompanies the final elaboration of the
complex or that, within the complex, at the most residues
P14 to P6 of the RCL are inserted into
-sheet A. These conclusions limit drastically the possible locations
of the defeated protease within the complex.
 |
INTRODUCTION |
Serine protease inhibitors
(serpins)1 are mainly
composed of three
-sheets (A, B, and C) united by nine
-helices
(A-I); indeed, many are inhibitors that neutralize their target(s) by
forming a, stoichiometric, stable complex (1-3). Formation of the
stable complex involves the charge stabilizing system of the target
protease (4-8) and a surface loop of the inhibitor called the reactive center loop (RCL). The RCL connects strand 4 of
-sheet A to strand 1 of
-sheet C; it is exposed to solvent in the inhibitory serpins. By
analogy with protease substrates, the 20 amino acids constituting the
RCL are numbered Pn- ...
-P1-P'1- ... -P'n, where P1-P'1 is ultimately cleaved. The
mechanism of protease inhibition involves multiple steps that initiate
by the formation of a reversible association, converting to a stable
complex, ultimately split into regenerated enzyme and RCL-cleaved
(consumed) serpin (9-12). The RCL sustains a variety of conformations
(13-16). In antithrombin (AT) that is heparin-activated (17, 18) and
other inhibitory serpins such as
1-antitrypsin (also called
1-proteinase inhibitor; 19-21) or
1-antichymotrypsin (22), the
RCL is wholly exposed, whereas in the AT monomer, residue
P14 of the RCL disrupts
-sheet A (23-25). In latent AT,
an intact but non-inhibitory conformer (25), and in latent type-1
plasminogen activator inhibitor (16), residues P14 to
P3 of the RCL are completely inserted into
-sheet A,
constituting an additional, sixth, strand. The same conversion from a
five- to six-stranded
-sheet A occurs in the inhibitory serpins,
following cleavage of the RCL (14, 26).
To date, no x-ray analysis of a serpin-protease complex has been
reported; thus, its structure remains largely hypothetical. Based on
functional studies of serpin variants and immunochemical investigations, a number of reports nevertheless suggest that, in the
stable complex, the RCL is inserted into
-sheet A. Antibodies have
been characterized that fail to react with native serpin, but recognize
binary complexes with a synthetic tetradecapeptide corresponding to
residues P14 to P1 of the RCL, as well as
consumed inhibitors (27-33). Thus, antibodies revealed that insertion
of the RCL in
-sheet A exposes neoepitopes that are not present in
the intact inhibitor. The same neoepitopes being exposed in stable
serpin-protease complexes led to the conclusion that, during trapping
of the protease, the RCL inserts at least in part into
-sheet A. Convincing evidence also suggests that the protease translocates away
from the site at which initial attack occurs. Wright and Scarsdale (34)
even proposed that the enzyme ends in a location almost opposite to
that of the RCL in intact serpins, i.e. that, in the stable
serpin-protease complexes,
-sheet A is a six-stranded sheet.
Stratikos and Gettins (35) demonstrated that, at least 21 Å separates
position of the protease in the initial (reversible) Michaelis complex
from that in the (virtually irreversible) final complex. Modeling
considerations suggest a range of plausible stable structures: from
full insertion of the RCL with upside-down translocation of the
defeated protease, to limited RCL insertion (up to the P9
residue) with concomitant stacking of the target on the F-helix.
Following cross linking experiments with stable complexes of type-1
plasminogen activator inhibitor, Wilczynska et al. (36)
favored stacking on the F-helix, and partial rather than complete
insertion of the cleaved RCL.
We report herein characterization of a monoclonal antibody (12A5) that
provides definite insight into the structure of the stable AT-protease
complex; unless AT was unfolded, the antibody failed to react with any
AT monomers, while endorsing stable complexes with thrombin or factor
Xa and binary complex with a short peptide derived from the RCL. The
unexpected location of the neoepitope, on the distal part of
-sheet
A, has several fundamental implications. The conformations of
-sheet
A and/or of the F-helix in the stable complex must simultaneously
differ from the five- and six-stranded structures of native and
RCL-cleaved AT, respectively. Steric hindrance considerations also
limit drastically the possible topologies of the complex.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human thrombin and its S195A variant were
prepared as described previously (8). Factor Xa was purchased from ERL
(South Bend, IL). Porcine pancreatic elastase (type IV) and bovine
serum albumin (BSA, protease-free) were from Sigma (St-Quentin
Fallavier, France), as well as mouse AT and bovine, sheep, porcine,
rabbit, and chicken plasma. Standard, unfractionated heparin (Heparin Choay) and pentasaccharide with high affinity for AT
(M, 1714) were from Sanofi-Winthrop (Gentilly, France).
Peptide P14-P9 derived from the RCL of AT
(Ac-SEAAAS) was synthesized by Altergen (Schiltigheim, France).
Preparation and Characterization of the Various AT
Conformers--
Human AT was purified from citrated frozen plasma
essentially according to McKay (37) by affinity chromatography on
heparin-Sepharose (Pharmacia, St-Quentin-en-Yvelines, France), followed
by anion-exchange chromatography on a Mono-Q column (Pharmacia). AT
cleaved by porcine pancreatic elastase was prepared by incubating AT (5 µM) with 50 nM enzyme for 4 h at
37 °C in 50 mM Tris-HCl, pH 8.5, containing 0.15 M NaCl and 0.1% polyethylene glycol
(Mr 8000; w/v). The resulting C-terminal
fragment was isolated by transfer onto polyvinylidene difluoride
membrane after denaturing polyacrylamide gel electrophoresis (15%
acrylamide), and analyzed by N-terminal sequencing (Biotechnology Department, Institut Pasteur, Paris, France). Two sites of cleavage were identified: Val389-Ile390 and
Ile390-Ala391, corresponding to the
P5-P4 and P4-P3
residues of the RCL, respectively. Identical cleavages are obtained
following incubation of AT with human neutrophil elastase (38).
Thrombin-cleaved AT was prepared by incubating AT (7 µM)
with thrombin (0.1 µM) for 3 h at 37 °C in 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl and
trace amounts of SDS (0.02%, w/v; Refs. 39 and 40). Thrombin was
neutralized by the addition of 1 mM phenylmethylsulfonyl
fluoride (Sigma). Traces of SDS and excess phenylmethylsulfonyl
fluoride were removed by extensive dialysis against 50 mM
Tris-HCl, pH 7.5, containing 0.15 M NaCl. Denaturing
polyacrylamide gel electrophoresis (Fig. 1) suggested that in these
conditions AT was fully cleaved by thrombin, at a single site. In
addition to the N terminus of AT, N-terminal sequencing of the reaction
mixture revealed a single sequence starting with the P'1
residue of the RCL, indicating that thrombin cleaved a single bond in
AT, between the P1 and P'1 residues of the RCL.
AT cleaved by porcine pancreatic elastase or by thrombin were
indistinguishable by polyacrylamide gel electrophoresis analysis (Fig.
1) and had a similar affinity for heparin (i.e. reduced
compared with native AT); both eluted at about 0.3 M NaCl on heparin-Sepharose column. Latent AT was prepared according to
Wardell et al. (41). Briefly, AT (1.7 µM) was
incubated for 18 h at 60 °C in 10 mM Tris-HCl, pH
7.5, containing 0.35 M sodium citrate. Following
incubation, the mixture was dialyzed against 10 mM
Tris-HCl, pH 7.5, and latent AT was recovered by heparin-Sepharose chromatography and anion-exchange chromatography on Mono Q column, both
developed with a linear gradient from 0.0 to 0.5 M NaCl; latent AT eluted at 0.3 and 0.2 M NaCl, respectively. The
concentration of all AT monomers was estimated assuming an absorption
coefficient of 3.8 × 104 M
1
cm
1 at 280 nm (
%=6.5 cm
1). Stable
complexes of AT with thrombin or factor Xa were prepared by incubating
AT (5 µM) for 2 h at room temperature with thrombin (5 µM) or for 4 h with factor Xa (5 µM) in 50 mM Tris-HCl, pH 7.5, containing
0.15 M NaCl and 0.1% polyethylene glycol
(Mr 8000; w/v). Virtually no monomer of AT
(either native or RCL-cleaved) could be seen by polyacrylamide gel
electrophoresis analysis in the presence of SDS (Fig. 1); therefore,
concentrations of the complexes were assumed to be that of the added AT
(5 µM). Reversible complex of AT with S195A thrombin was
prepared by mixing AT (2 µM) with S195A thrombin (2 µM) in the presence of heparin (1 unit/ml); association
was presumed to be almost instantaneous (8), and concentration of the
reversible complex was assumed to be that of the added AT. Formation of
a binary complex with the P14-P9 peptide
derived from the RCL was carried out according to Schulze et
al. (28) and Chang et al. (42), by incubation of AT (20 µM) and peptide (2 mM) in 50 mM
Tris, pH 8.0, containing 50 mM NaCl. Binary complex was
used immediately after a 60-min incubation at 37 °C: when less than
50% AT formed a complex and no polymerization of AT had yet occurred
(Fig. 1). Concentration of the binary
complex was assumed to be 40% that of the total amount of AT.

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Fig. 1.
Polyacrylamide gel electrophoresis of the
various AT conformers used. Left panel, electrophoresis
in denaturing conditions of: native AT (lane 1),
AT cleaved by porcine pancreatic elastase (lane
2), AT cleaved by thrombin (lane 3),
thrombin (lane 4), complex of AT with thrombin
(lane 5), factor Xa (lane
6), and complex of AT with factor Xa (lane
7). Right panel, electrophoresis in
non-denaturing conditions of: native AT (lane 1),
latent AT (lane 2), and AT partially in complex
with the P14-P9 peptide derived from the RCL
(lane 3).
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Preparation and Characterization of the Monoclonal
Antibodies--
Murine monoclonal antibodies directed against human AT
were selected, isolated and characterized by standard procedures (43). Four 6-week-old BALB/c females were immunized by subcutaneous injection
of 20 µg of AT in complete Freund's adjuvant followed 3 weeks later
by a further subcutaneous injection of 20 µg of AT in incomplete
Freund's adjuvant. One hundred days later, mouse received 8 µg of AT
subcutaneously and 12 µg of AT intravenously. One day prior to the AT
injection, and for 5 days, mice also received a daily intraperitoneal
injection of 20 µg of pentasaccharide. Three days after the final
injection, spleen cells were fused with the mouse myeloma cell line
P3-X63-Ag.8.653 by using 50% polyethylene glycol 1540, and
cultured in hypoxanthine/aminopterin/thymidine media. Positive
clones were detected by enzyme-linked immunosorbent assay (ELISA);
Maxisorp microplates from Nunc (Polylabo, Strasbourg, France) were
coated overnight at 4 °C with 5 µg/ml AT in 0.1 M Na2HCO3/NaH2CO3, pH
9.5, and washed three times in 20 mM
Na2HPO4/NaH2PO4, pH
7.4, containing 0.15 M NaCl (PBS) and 0.5% Tween 20 (v/v). Residual sites were blocked for 1 h with 1 mg/ml BSA in PBS, and microplates were washed as above. Each hybridoma supernatant to be
tested was added to one of the wells of the microplate and allowed to
bind for 1 h at room temperature. After washing, a peroxidase-conjugated goat anti-mouse antibody (Bio-Rad,
Ivry-sur-Seine, France) was added and allowed to bind for 1 h at
room temperature. Following a final wash, ELISA was developed by adding
150 µl of orthophenylenediamine (0.5 mg/ml; Bio-Rad) in 0.1 M sodium citrate, pH 5.5, containing 0.3%
H2O2 (v/v). The reaction was stopped after 30 min by the addition of 50 µl of H2SO4
(12.5%, v/v), and the absorbance at 490 nm was recorded. Selected
clones were grown in pristane-primed BALB/c mice, and antibodies
purified from the ascitic fluid by affinity chromatography on protein
A-Sepharose (Pharmacia). IgG appeared pure by denaturing polyacrylamide
gel electrophoresis analysis and were stored at
80 °C after
dialysis against PBS. The subclass was determined with a commercial kit (Amersham, Les Ulis, France): monoclonal 12A5 described in this study
was typed as IgG2a.
Determination of the AT Conformers Recognized--
To identify
the conformers of AT recognized by the monoclonal antibodies, a
sandwich ELISA was designed in which microplates were coated with
purified IgG (5 µg/ml). Each AT conformer, diluted in PBS containing
0.5% Tween 20 (v/v) and 1 mg/ml BSA, was added to one of the wells of
the microplate and allowed to bind for 2 h at room temperature.
Sandwich ELISA was developed with a peroxidase-conjugated goat IgG
directed against human AT (Sanofi Research, Montpellier, France). In
assays with S195A-AT reversible complexes, 1 unit/ml heparin was
present during the binding step, and all buffers included 0.1 unit/ml
heparin to prevent release throughout the washing steps. Ability of the
S195A-AT complexes to be retained on a microtiter plate was assessed by
the use of a commercial kit designed to detect plasma thrombin-AT
complexes (Enzygnost TAT micro; Behring, Marburg, Germany). A
microplate, coated with a polyclonal antibody directed against thrombin
was incubated with various amounts of S195A-AT complexes and developed
with a polyclonal antibody directed against AT, according to the
manufacturer's instructions except that 0.1 unit/ml heparin was added
in all buffers.
Epitope Mapping--
Customized AT peptide sets were prepared by
Chiron Mimotopes Peptide Systems (Clayton, Victoria, Australia) as
biotinylated peptides linked to a cleavable polyethylene pin (44). A
four-amino acid spacer (GSGS) separated the N terminus of each peptide
from the pin, except for the peptide encompassing the first 14 amino acids of AT where the spacer was coupled to the C terminus. Binding assays were performed according to the supplier's instructions. Briefly, 100 µl of streptavidin (5 µg/ml in water) was added to each well of a microplate and evaporated to dryness at 37 °C. Biotinylated peptides (about 1 µM in PBS containing 1 mg/ml BSA) were added and incubated for 1 h at room temperature.
Monoclonal antibody (0.1 µg/ml) in PBS containing 0.5% Tween 20 (v/v) and 1 mg/ml BSA was incubated for 2 h at room temperature.
Washing and development were otherwise performed as described above for the ELISA, using the peroxidase-conjugated goat anti-mouse IgG.
Immunoblotting--
Immunoblotting of the various AT conformers
(after polyacrylamide gel electrophoresis in denaturing conditions) was
performed on nitrocellulose membrane (Bio-Rad) essentially as described previously (45). Incubation and washing were all completed at room
temperature in 50 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl and 0.5% Tween 20 (v/v). The membrane was saturated
with nonfat dry milk (3% w/v), washed, and incubated for 1 h with
monoclonal antibody 12A5 (0.5 µg/ml). After washing, the membrane was
incubated for 1 h with an alkaline phosphatase-conjugated goat
anti-mouse IgG (Bio-Rad). The immunoblot was further washed, and
developed by adding a phosphatase substrate solution consisting of 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.3 mg/ml nitro blue
tetrazolium in 0.1 M Tris, pH 9.5, containing 0.5 mM MgCl2.
Inhibition Kinetics--
The influence of monoclonal antibody
12A5 on thrombin and factor Xa inhibition by AT was studied in the
absence and in the presence of heparin, essentially as described
previously (46, 47). Assays were performed at 37 °C in 50 mM Tris-HCl, pH 7.6, containing 0.15 M NaCl,
and 0.1% polyethylene glycol (Mr 8000; w/v); 1 mg/ml BSA was included in the assay of factor Xa inhibition. Without
heparin, estimates of the association rate constant
(kon), in the presence of 1 µM
12A5 (or control IgG), were obtained from kinetic experiments performed
in microplates, under pseudo first-order conditions (20-100
nM AT with 2 nM thrombin, and 150-300
nM AT with 15 nM factor Xa). First order rate
constants were estimated by non-linear regression analysis of the
residual activities versus time (up to 90 min), and
kon values deduced from the linear plot of the
first order rate constants as a function of AT. The
kon values in presence of heparin (1.4 units/ml), were estimated by analysis of data from progress-curve
kinetics also completed in pseudo first-order conditions (0.2-0.6
nM AT with 10 pM thrombin, and 1-2
nM AT with 100 pM factor Xa). Inhibition of
thrombin was initiated by its addition to a microtiter well containing
0.2 µM 12A5 (or control IgG), AT at various
concentration, and 100 µM
H-D-Phe-pipecolyl-Arg-p-nitroanilide (S-2238,
Biogenic, Montpellier, France). Before initiating factor Xa inhibition,
the release of p-nitroaniline from 400 µM
benzyl-CO-Ile-Glu-(
-OR)-Gly-Arg-p-nitroanilide (S-2222,
Biogenic) by factor Xa was monitored for 10-15 min, until a steady
state velocity of about 0.2 µM min
1 was
reached. The inhibition reaction was initiated by the addition of a
mixture of 0.2 µM 12A5 (or control IgG) and AT at various concentrations. The release of p-nitroaniline was monitored
for up to 3 h using a Lambda 14 Perkin Elmer spectrophotometer,
but only data corresponding to less than 10% substrate hydrolysis were
used in the analysis. Estimates of the kon
values were obtained by fitting data to the equation for slow-binding
inhibition and corrected for the competition introduced by the substrate.
 |
RESULTS |
12A5, a Monoclonal Antibody That Distinguishes AT-Protease
Complexes from Native, Latent, and RCL-cleaved AT--
AT may adopt at
least four conformations: native, heparin-activated, latent, and
RCL-cleaved. A fifth conformation is likely to occur when AT is trapped
in a complex with one of its targets (thrombin or factor Xa), but the
precise structure of AT within this complex remains largely unknown. In
an attempt to probe the elusive conformation of trapped AT, we prepared
a panel of monoclonal antibodies; 12 were selected, because they
recognized AT that had been coated on a microplate. To determine which
conformer of AT was recognized, the monoclonal antibodies were coated
onto a microplate, then AT (native, RCL-cleaved, or in complex with thrombin) was added, and sandwich ELISA developed with a polyclonal antibody directed against AT. Two monoclonal antibodies retained our
attention, because they did not react with native or RCL-cleaved AT,
while giving a strong signal with complexes; the other monoclonal antibodies reacted with all three forms of AT, indicating that selectivity of the former was not due to an experimental artifact. Thus, even though AT-protease complexes were not included in the immunization mixture, all the antibodies reacted with AT in complex. Although surprising at first, we reasoned that complexes might have
formed with endogenous mouse proteases, and that a neoepitope inaccessible in the native protein might be exposed in partially denatured AT. To delineate more precisely the specificity of one of the
monoclonal antibodies (12A5), we evaluated its affinities for native,
latent, and heparin-activated AT, for AT with the RCL cleaved by
thrombin or porcine pancreatic elastase, for AT in stable complex with
thrombin or factor Xa, and for AT in complex with a peptide derived
from the P14-P9 residues of its RCL. AT in
complex with either thrombin or factor Xa bound to 12A5 in a
dose-dependent fashion, as did the binary complex of AT
with the P14-P9 peptide, although to a lesser
extent, but binding of the other AT conformers remained undetected at
concentrations as high as 1 µM (Fig.
2). In contrast, 12A5 recognized all
forms of AT following polyacrylamide gel electrophoresis in the
presence of SDS and immunoblotting, even after reduction of the
disulfide bridges with
-mercaptoethanol. Thus, a motif recognized by
12A5, inaccessible in native, heparin-activated, latent, and
RCL-cleaved AT, was exposed in denatured and target-complexed AT. Taken
together, these data suggested that steric hindrance, rather than a
need for a specific conformation, limited the ability of 12A5 to
interact with native or RCL-cleaved AT.

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Fig. 2.
Antibody 12A5 recognizes only complexes of AT
with thrombin, factor Xa, or with the P14-P9
peptide derived from the RCL. Antibody 12A5 was coated onto
microtiter wells, and AT in complex with thrombin ( ), AT in complex
with factor Xa ( ), native AT (×), latent AT ( ),
heparin-activated AT ( ), AT cleaved by porcine pancreatic elastase
( ), AT cleaved by thrombin ( ), or AT in complex with the
P14-P9 peptide derived from the RCL ( ) was
added and allowed to bind for 2 h. Sandwich ELISA was developed
with a peroxidase-conjugated polyclonal IgG directed against AT.
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12A5 Recognizes Pentapeptide DAFHK on the Penultimate Strand of
-Sheet A--
As 12A5 recognized unfolded AT after reduction of the
disulfide bonds, it was reasonable to expect that the neoepitope was a
contiguous sequence of amino acids. We scanned two sets of biotinylated peptides, covering the entire sequence of AT. The first set simply divided AT sequence into 31 peptides that each were 14 amino acids in
length, starting with His1 of AT; the second set divided AT
sequence into 30 peptides (each also 14 amino acids in length) but
starting with Cys8 of AT. Thus, each peptide overlapped 2 peptides of the alternate set by 7 amino acids (except peptides 1 and
31 of the first set). Peptides were individually linked to the well of
a microtitration plate, and incubated with 12A5. ELISA was developed
with a peroxidase-conjugated goat anti-mouse antibody. Only two
peptides of the whole bank were able to bind 12A5: GRDDLYVSDAFHKA and
SDAFHKAFLEVNEE, suggesting that the neoepitope included the shared
motif SDAFHKA. Consistent with this hypothesis, 0.5 µM
thrombin-AT displaced 50% of the binding of 10 nM 12A5 to
peptide SDAFHKA coated on a microplate. Similarly, 0.2 µM
peptide SDAFHK displaced 50% of the binding of 2 nM
thrombin-AT to 12A5 coated on a microplate. To refine the neoepitope,
we determined the shortest sequence that 12A5 could recognize by
preparing a third set of peptides that consisted of successive,
discrete, amino acid exclusions from either ends of peptide VSDAFHKAF.
The shortest sequence recognized was DAFHK (Table
I). The importance of each amino acid
within the pentapeptide was evaluated by alanine scanning: aspartate,
phenylalanine, and lysine were strictly required for 12A5 binding; in
contrast, alanine in position 2 and histidine in position 4 could be
substituted without affecting the binding capability of 12A5 (Table I).
In the structure of AT, motif DAFHK is on the penultimate strand of
-sheet A, and is not accessible to solvent (Fig.
3). The identity of the neoepitope was
further confirmed by testing the reactivity of 12A5 with AT from
various species. Motif DAFHK is conserved in human, bovine, sheep,
porcine, and rabbit AT; all were recognized by 12A5 in immunoblotting
analysis. In chicken AT, residue 366 is a glutamate instead of an
aspartate. Consistent with the hypothesis that Asp366 was
strictly required, 12A5 failed to recognize chicken AT (Fig. 4). Unexpectedly, mouse AT also contains
the motif DAFHK and was recognized in immunoblotting, implying that
12A5 was an autoantibody. However, mammalian synthesis of an
autoantibody directed against a cryptic region of a self-protein is not
implausible; in fact, Nordling and Björk (33) have characterized
a rabbit serum that recognizes the sequence VDLFSP shared by rabbit and
human AT.
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Table I
Identification of 12A5 epitope
Table shows binding of 12A5 to peptides derived from sequence
Val364 to Phe372 of AT. Biotinylated peptides (linked
to a microplate) were considered as able to grasp 12A5 (+) when signal
was in excess of 8-fold that of the background. The minimum sequence
recognized was DAFHK; within this sequence, only D, F, and K were found
to be critical.
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Fig. 3.
Location of neoepitope DAFHK within the
native and latent conformers of human AT. Stereo views of native
(upper panel) and latent (lower
panel) conformers of AT drawn using Molscript (version 3.1).
Coordinates were obtained from the Protein Data Bank (access code
1ANT). Views are centered on -sheet A, and the location of the
F-helix is indicated, as are the side chains of the residues critical
for 12A5 binding (Asp366, Phe368, and
Lys370). Consistent with the lack of recognition of both
conformers by 12A5, Phe368 is partly masked to solvent by
the loop connecting the F-helix to strand 3 of -sheet A.
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Fig. 4.
Antibody 12A5 is an autoantibody.
Immunoblotting of sheep (lane 1), rabbit
(lane 2), porcine (lane 3),
and bovine plasma (lane 4); AT from all species
share the motif DAFHK and were recognized by 12A5. In chicken AT
(lane 5), residue 366 is a glutamate (instead of
an aspartate); chicken plasma was not recognized by 12A5. Surprisingly,
purified mouse AT (lane 6) was recognized by
12A5. Mouse AT contains a DAFHK sequence, implying that immunized mice
synthesized an autoantibody against a cryptic region of their own
protein. The last lane was obtained with purified human AT; the small
lower band corresponds to -AT (45).
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The Conformational Change Unveiled by 12A5 Is Subsequent to the
Step Controlled by Ser195 of the Protease--
To achieve
formation of a stable complex, serpin-protease interactions involve
multiple steps, but in the path of AT inhibition, only the very last
concerns the catalytic serine of the protease. S195A thrombin is an
inactive variant, in which the catalytic serine has been replaced by
alanine (8). S195A binds to AT with a kon value
similar to that of thrombin, indicating that Ser195 of
thrombin is not involved in the rate-limiting step of complex formation. The main difference between thrombin and S195A actually resides in the rate of complex dissociation; in contrast to the thrombin-AT complex, the S195A-AT complex is reversible
(KI = 3 nM with heparin, 3 µM otherwise). Unless AT was denatured, the motif DAFHK
appeared accessible to 12A5 only when AT was in complex with a peptide
or a target. Thus, the combination of S195A and 12A5 constituted a
remarkable tool to probe the concluding step of thrombin-AT complex
formation; if the particular folding of AT unveiled by 12A5 takes place
after formation of the initial complex, but before the irreversible
step, AT bound to S195A must react with 12A5. On the contrary, if
neoepitope exposure is concomitant with final stabilization of the
complex, then motif DAFHK must remain invisible to 12A5, whether or not
S195A adheres to AT. The high KI value of AT for
S195A in the absence of heparin precluded study of 12A5 binding to the
reversible complex, but saturation experiments were feasible in the
presence of heparin. Hence, saturating amounts of S195A-AT complexes
were not retained on a microplate coated with 12A5 (Fig.
5). This observation implied that
neoepitope display was independent from initial binding of AT to
thrombin; it was instead concomitant (or subsequent) to the catalytic
step involving Ser195. Consistent with this hypothesis,
12A5 had no detectable influence on thrombin or factor Xa inhibition by
AT; kon values were virtually identical, whether
or not the target had been preincubated with saturating concentration
of 12A5, and that reaction proceeded in the presence or not of heparin
(Table II and Fig.
6). In fact, mice producing 12A5 were not
abnormally ill, suggesting that autoantibody did not interfere with
normal hemostasis, at least until neoepitope DAFHK was exposed
(i.e. after formation of the stable complex). In this model,
the autoantibody would not be harmful; the only consequence might have
been a faster clearance of the protease-AT complexes.

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Fig. 5.
Antibody 12A5 fails to recognize the
reversible complex of AT with S195A thrombin. Binding of the
stable thrombin-AT complex ( ) and of the reversible S195A-AT complex
( ) to microtiter wells coated with 12A5. Sandwich ELISA was
developed with a peroxidase-conjugated goat IgG directed against human
AT. Lack of recognition does not originate from S195A-AT complex
dissociation during the washing steps; middle curve ( ) represents
binding of the S195A-AT complex to microtiter wells coated with a
rabbit IgG directed against thrombin and revealed by a
peroxidase-conjugated rabbit IgG directed against AT. Saturation
suggests that washing does not dissociate the S195A-AT complex.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Association rate constants (kon) for thrombin and factor Xa
inhibition by AT, with or without 12A5 antibody, and with or without
heparin
Estimates of kon (in M 1
s 1) are given together with their standard error (in
parentheses). With or without 12A5, kon values were
similar, suggesting that 12A5 did not bind to AT until a stable complex
was formed.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Antibody 12A5 does not influence thrombin and
factor Xa inhibition by AT. Progress curves of thrombin inhibition
(upper panel) and factor Xa inhibition
(lower panel) in the presence of heparin.
Reactions were performed in the presence of 12A5 antibody ( ) or in
the presence of a monoclonal antibody that does not recognize AT,
thrombin, or factor Xa ( ). Solid lines were
obtained by non-linear regression analysis of the data according to the
equation for slow binding inhibition.
|
|
 |
DISCUSSION |
Our data establish that AT in complex with thrombin or factor Xa
differs from all conformers of known structure (native,
heparin-activated, latent, or RCL-cleaved); they also imply that
protease permits access to neoepitope DAFHK within the complex.
In none of the structures of AT available (18, 23, 25, 26) is the motif
DAFHK entirely accessible to solvent. The side chains of
Ala367 and His369 (dispensable for 12A5
binding) are internal; those of Asp366, Phe368,
and Lys370 (critical for 12A5 binding) are directed toward
the exterior, but in the native conformer they are partly hidden from
solvent, except for Asp366 (Fig. 3). In latent AT,
Asp366 contacts Val389 and Leu395
(P4 and P'2 residues of the RCL, respectively).
Most remarkably, the side chain of Phe368 neighbors segment
Ile198-Pro203 of the loop connecting the
F-helix to strand 3 of
-sheet A, both in the native and latent
conformers of AT. Phe368 actually occupies a hydrophobic
pocket lined by Val201, Ile202,
Pro203, and Ala206; somehow, this pocket must
open for 12A5 to bind. Lys370 also neighbors segment
Ile198-Pro203; it is ion-paired to
Asp200. Molecular contacts in the RCL-cleaved conformer of
bovine AT are similar to those occurring in latent (human) AT. Thus,
this neoepitope is constrained in native, as well as in latent and RCL-cleaved AT, and it is reasonable to predict that these constraints prevented 12A5 binding; conversely, formation of the stable complexes recognized by 12A5 must have released these constraints. The
conformational change that accompanies complex formation and allows
exposure of the neoepitope could be so dramatic, that the overall
folding of AT has collapsed. This hypothesis would be consistent with our observation that 12A5 also recognized unfolded AT (i.e.
blotted on nitrocellulose membrane or adsorbed on plastic wells).
However, such a model would be difficult to reconcile with the dynamics of protease trapping by a serpin; in general, bonds are cleaved more
efficiently in denatured substrates than in folded proteins (48). Thus,
if formation of an initial Michaelis complex triggers gross disruption
of AT folding, it would be expected that AT is a good substrate rather
than an inhibitor. More satisfactory is a model in which the overall
folding of AT is preserved: failure of 12A5 to react would originate
from a steric hindrance that complex formation releases. Epitopes have
been characterized that are recognized in the latent, RCL-cleaved, and
complexed conformers, not in the native serpin; hence, RCL-cleaved and
native conformers share a common overall folding, implying that limited
conformational change indeed can trigger exposure of a neoepitope (27,
33, 49). Two limited alterations in the structure of AT would permit motif DAFHK to access solvent: partial disruption of
-sheet A and/or
motion of the loop connecting the F-helix to strand 3 of
-sheet A
(Fig. 3).
A number of studies suggest that several events, including formation of
the stable complex, dramatically modify the structure of
-sheet A in
serpins. In type-1 plasminogen activator inhibitor, for instance, a
temperature-dependent transition exposes the penultimate strand of
-sheet A, as attested by its susceptibility to non-target proteases (50); at 0 °C, the native (inhibitory) conformer is cleaved within motif QALQK (i.e. within the region
homologous to neoepitope DAFHK of AT); at 37 °C, the motif resists
hydrolysis. The main structural rearrangement that seems to accompany
complex formation consists of an insertion of the N-terminal (P) side of the RCL into
-sheet A, but it is unclear which of the
P14 to P1 residues are concerned (49, 51-54).
It is likely that insertion is subsequent to the release by the
protease of the C-terminal (P') side of the cleaved RCL (11, 55),
whereas the P1 residue remains attached to the charge
stabilizing system of the protease. Insertion of peptides with the same
sequence as the RCL also causes conformational changes in AT; there,
the extent of the insertion triggers various consequences, suggesting
that many conformations are achievable (29, 42, 56, 57). Structural
data obtained by x-ray diffraction (18, 25), fluorescence spectra data
on the S380W variant of AT (17), immunochemical data (58), and susceptibility of the P1 arginine to chemical modification
(59) all suggest that at least the P14 residue of the RCL
is inserted in native (inhibitory) AT; this conformer was not
recognized by 12A5 antibody. Insertion of a 7-amino acid peptide,
homologous to residues P14-P8 of the RCL,
induces a slow polymerization of AT (42), but insertion beyond the
P8 residue, prevents polymerization (57). Neoepitope was
recognized by 12A5 in the binary complex of AT with a peptide
homologous to residues P14-P9 of its RCL. On
the other hand, the neoepitope was no longer recognized by 12A5 in the
conformer of AT having the RCL cleaved at the
P5-P4 bond (i.e. with the RCL
inserted up to the P5 residue), nor in AT cleaved at the
P1-P'1 bond by thrombin (i.e. with
the RCL inserted up to the P4 residue; Ref. 26). Finally,
when a synthetic tetradecapeptide homologous to residues
P14 to P1 of the RCL is locked in
-sheet A,
AT is a substrate rather than an inhibitor (30, 31). Thus, insertion of
not less than two, but no more than nine, residues in
-sheet A
induced a particular conformation in AT that allowed 12A5 to bind the
motif DAFHK. This neoepitope was also accessible to 12A5 in the stable
complexes with thrombin or factor Xa. Therefore, it is tempting to
conclude that AT in complex with either a target or the
P14-P9 peptide share similar structural
rearrangements: absent from the five- as well as from the six-stranded
-sheet A conformers. Combined with the assumption that part of the
RCL occupies
-sheet A within the stable complex, the inference is that insertion concerns at least the P14-P13
residues of the RCL, at most the P14 to P6 segment.
An alternative to partial disruption of
-sheet A would be that
failure of 12A5 to react originates from a steric hindrance that is
unrelated to the conformation of the penultimate strand of
-sheet A. Unless it is masked, the motif DAFHK would be recognized by 12A5 in the
five- as well as in the six-stranded conformers: structure of
-sheet
A would not be the limiting factor per se. Displacement of
the loop connecting the F-helix to strand 3 of
-sheet A would
authorize neoepitope recognition by 12A5. Somehow, insertion of the RCL
into
-sheet A (as observed in latent and RCL-cleaved serpins)
requires that the connection to the F-helix moves: otherwise, the RCL
would have to sneak into a very narrow passage underneath the
connecting loop. Fairly limited motion of this surface loop would
suffice to clear access to motif DAFHK. Once the RCL is inserted, the
surface loop could return to a location similar to that which it
occupied initially, with the result that the neoepitope is masked again
in the conformer with fully inserted RCL. It is unlikely that surface
loop moves alone; comparison of RCL-cleaved and native serpins suggests
that strands 1-3 of
-sheet A, the F-helix, and the connecting loop
constitute a mobile block that shifts relative to the remainder of the
structure (60). Considering that the neoepitope was masked in both the
native and RCL-cleaved conformers of AT, the inference is that the
motif DAFHK is accessible to 12A5 only in the utmost distorted position of the mobile block, and that this distorted conformation is similar in
the stable complex with a target. Consistent with this hypothesis, studies of a functional epitope in type-1 plasminogen activator inhibitor and of natural variants of AT or C1 inhibitor pointed out an
unexpected role of the F-helix and of its connecting loop to the
-sheet A (61-63).
The observation that motif DAFHK remained accessible within the complex
AT-protease restricts the possible topologies of the complex (34-36).
One of the proposals is that the enzyme ends in a location almost
opposite to the RCL in intact serpins. This model is compatible with
our data, as the protease would not cover motif DAFHK. However, it is
difficult to reconcile the hypothesis that
-sheet A is a
six-stranded structure in the stable complex with our observation that
neoepitope is not exposed following full insertion of the cleaved RCL.
Thus, our data imply that if the protease rotates fully around the
serpin, then either
-sheet A is grossly unfolded within the complex
(i.e. it is neither a five nor a six-stranded structure), or
the F-helix has moved away to release motif DAFHK. Assuming on the
contrary that the overall folding of AT is preserved, and that the
cleaved RCL inserts partially into
-sheet A, the complexed protease
cannot rotate further than the F-helix around AT (i.e. about
half-way), as it would otherwise cover the motif DAFHK. In this
respect, it is interesting to note that 12A5 was incapable of even
slowing down the inhibition reaction, and did not recognize the complex
of AT with the S195A thrombin mutant; the lack of adverse effects on AT
function suggests that only the very last step along the path of the
reaction triggered exposure of the neoepitope. It is likely that this
step is subsequent to the cleavage of the RCL.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Jean François Gaucher from
the Université Paris V for Fig. 3, Dr. Paul Hopkins from the
Gladstone Institute of Cardiovascular Disease San Francisco for helpful
discussion, and Gregory Chang (Inserm U428) for careful reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by INSERM, France.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: INSERM U428,
Faculté de Pharmacie, Université Paris V, 4 Av. de
l'Observatoire, 75270 Paris Cedex 06, France Tel.: 33-1-53-73-98-28;
Fax: 33-1-44-07-17-72; E-mail: lebonnie{at}infobiogen.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
serpin, serine
protease inhibitor;
AT, antithrombin (previously called antithrombin III);
RCL, reactive center loop (also called reactive site loop);
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked immunosorbent assay.
 |
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