Tài liệu Báo cáo khoa học: Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents pptx

Mapping of the epitope of a monoclonal antibody protecting
plasminogen activator inhibitor-1 against inactivating agents
Julie S. Bødker, Troels Wind, Jan K. Jensen, Martin Hansen, Katrine E. Pedersen and Peter A. Andreasen
Laboratory of Cellular Protein Science, Department of Molecular Biology, University of Aarhus, Denmark
Plasminogen activator inhibitor-1 (PAI-1) belongs to the
serpin family of serine proteinase inhibitors. Serpins inhibit
their target proteinases by an ester bond being formed
between the active site serine of the proteinase and the P
1
residue of the reactive centre loop (RCL) of the serpin, fol-
lowed by insertion of the RCL into b-sheet A of the serpin.
Concomitantly, there are conformational changes in the
flexible joint region lateral to b-sheet A. We have now, by
site-directed mutagenesis, mapped the epitope for a mono-
clonal antibody, which protects the inhibitory activity of
PAI-1 against inactivation by a variety of agents acting on
b-sheet A and the flexible joint region. Curiously, the epitope
is localized in a-helix C and the loop connecting a-helix I and
b-strand 5A, on the side of PAI-1 opposite to b-sheet A and
distantly from the flexible joint region. By a combination of
site-directed mutagenesis and antibody protection against an
inactivating organochemical ligand, we were able to identify
a residue involved in conferring the antibody-induced con-
formational change from the epitope to the rest of the
molecule. We have thus provided evidence for communi-
cation between secondary structural elements not previously
known to interact in serpins.
Keywords: cancer; cardiovascular disease; monoclonal
antibody; protease; serpin.
The serpins constitute a protein family of which the best
characterized members, including a

1
-proteinase inhibitor,
antithrombin III, and plasminogen activator inhibitor-1
(PAI-1), are inhibitors of serine proteinases implicated in
processes such as blood coagulation and turn-over of
extracellular matrix. Of decisive importance for the
inhibitory mechanism of serpins is the surface-exposed,
approximately 20-amino acid long reactive centre loop
(RCL) (see Fig. 1). Biochemical and biophysical evidence
has shown that the reaction between a serpin and its
target proteinase is initiated by formation of a reversible
docking complex in which the P
1
–P
1
¢ bond in the RCL
interacts noncovalently with the active site of the
proteinase [1]. In the locking step that follows, the P
1
–
P
1
¢ bond is cleaved [2,3] and the P
1
residue is coupled to
the active site serine of the proteinase by an ester bond
[4]. The N-terminal part of the RCL then becomes
inserted as strand 4 in b-sheet A (s4A) [5]. Because of the
covalent bond, the proteinase is translocated to the
opposite pole of the serpin [6–8], the active site becoming

distorted, the catalytic machinery inactivated, and the
completion of the catalytic cycle disabled [8–16], resulting
in formation of a stable covalently coupled complex of
1 : 1 stoichiometry (for reviews see [17–19]). The energy
needed for the proteinase distortion comes from stabi-
lization of the serpin in the ÔrelaxedÕ conformation by
insertion of the RCL into b-sheet A, as opposed to the
ÔstressedÕ, relatively unstable active conformation with a
surface-exposed RCL. Under some conditions, proteinase
distortion cannot keep pace with ester bond hydrolysis,
resulting in abortive complex formation, full cleavage of
the P
1
–P
1
¢ bond, insertion of the RCL into b-sheet A and
release of an active proteinase (for reviews see [17,20]).
Serpins following this alternative path are said to exhibit
substrate behaviour. Some serpins, including PAI-1 and
antithrombin III spontaneously assume an inactive,
relaxed, so-called latent state in which the intact RCL is
inserted into b-sheet A, after passage through the so-
called gate region between the s3C–s4C loop and the
s3B–hG loop (Fig. 1) [21,22].
RCL insertion is coupled to conformational changes in
the flexible joint region around a-helices D and E. The
flexible joint region of stressed, but not relaxed PAI-1,
binds to the N-terminal 44-amino acid long somatomedin
B domain of the M
r

70 000 glycoprotein vitronectin (VN)
[23,24], which thereby delays the latency transition of
PAI-1 (for a review see [20]). A few organochemical
compounds able to inactivate PAI-1 have been indentified,
including a group of negatively charged amphipathic
compounds like bis-ANS (4,4¢-dianilino-1,1¢-bisnaphthyl-
5,5¢-disulfonic acid) [11,25] and the diketopiperazine
derivative XR5118 ((3Z,6Z)-6-benzylidene-3-(5-((2-dimeth-
ylaminoethyl-thio)-2-thienyl)methylene-2,5-piperazinedione
Correspondence to J. S. Bødker, Department of Molecular Biology,
University of Aarhus, Gustav Wied’s Vej 10C, 8000 C Aarhus,
Denmark. Tel.: + 45 89425079, E-mail:
Abbreviations: bis-ANS, 4,4¢-dianilino-1,1¢-bisnaphtyl-5,5¢-disulfonic
acid; h, a-helix; RCL, reactive centre loop; HBS, Hepes buffered
saline; PAI-1, plasminogen activator inhibitor-1; s, b-strand;
S-2444, pyro-Glu-Gly-Arg-p-nitroanilide; uPA, urokinase-type
plasminogen activator; VN, vitronectin; wt, wild-type; XR5118,
((3Z,6Z)-6-benzylidene-3-(5-((2-dimethylaminoethyl-thio)-
2-thienyl)methylene-2,5-piperazinedione hydrochloride).
(Received 3 December 2002, revised 5 February 2003,
accepted 13 February 2003)
Eur. J. Biochem. 270, 1672–1679 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03523.x
hydrochloride) [11,26]. Their exact binding sites in PAI-1
remain to be established, but all available evidence is in
agreement with these compounds having overlapping, but
not identical, binding sites in the flexible joint region [27].
VN protects PAI-1 from inactivation by bis-ANS and
XR5118 [11,24,28]. These compounds do not bind to
relaxed PAI-1 [11]. Thus, there is bidirectional communi-
cation between the flexible joint region and the move-

ments of the RCL.
Among a large number of monoclonal antibodies
directed against PAI-1 raised since the mid-1980s, Mab-1
possesses a number of unique features. Mab-1 was raised
against latent PAI-1 purified from HT-1080 cells [29]. It
stabilizes PAI-1 against cold-induced substrate behaviour
in buffers with nonionic detergents [30]. As monitored by
proteolytic susceptibility, Mab-1 seems to induce con-
formational changes of the RCL, s5A, and the flexible
joint region [30,31]. In a recent study aimed at mapping
molecular interactions of PAI-1 by random mutagenesis,
Stoop et al. [32] identified residue Q58/74 as part of the
epitope for Mab-1 (a double amino acid numbering
system is used here, the first number following the
numbering system of Andreasen et al. [33], the second
number following the a
1
-antiproteinase inhibitor num-
bering system of Huber and Carrell [34]). Q58/74 is
localized in a-helix C (hC) (Fig. 1). Since it is localized
distantly from the secondary structural elements affected
by Mab-1, we hypothesized that a further characteriza-
tion of the epitope for Mab-1 might yield important
information about general aspects of serpin conforma-
tional changes.
Materials and methods
PAI-1
The cDNAs for wild-type (wt) and substituted human
PAI-1, extended at the N terminus with a His
6

-tag and a
recognition motif for heart muscle kinase, were produced
by standard methods in the Escherichia coli expression
vector pT7-PL [35]. Transformed E. coli BL21(DE3)-
pLysS cells from 1-L cultures, treated with 0.5 m
M
isopropyl thio-b-
D
-galactoside to induce PAI-1 expression,
were harvested by centrifugation (7000 g, 30 min), resus-
pended in 35 mL phosphate-buffered saline (10 m
M
Na
2
HPO
4
,140 m
M
NaCl pH 7.4), and disrupted by soni-
cation. The homogenates were centrifuged (10 000 g,
20 min), filtered (0.22 lm), supplemented with 2
M
NaCl
and 10 m
M
imidazole, and applied to a 5-mL Ni-NTA
column equilibrated in the same buffer further supple-
mented with 5% glycerol. PAI-1 was eluted with 200 m
M
imidazole. The eluted protein was subjected to gel

filtration on a Superdex 75 column (1.6 · 60 cm) equi-
librated in Hepes-buffered saline (HBS; 10 m
M
Hepes,
140 m
M
NaCl pH 7.4) supplemented with 5% glycerol
andNaCltoafinalconcentrationof1
M
. The procedure
routinely gave 10–15 mg PAI-1 per litre bacterial culture.
The preparations contained PAI-1 which was more than
95% pure as evaluated by SDS/PAGE and Coomassie
blue staining. N-terminal sequencing showed the expected
N terminus, i.e. (M)GSMGSHHHHHHGSRRASV
HH…, missing only the initiating M indicated in paren-
theses. The N-terminal extension did not affect the specific
Fig. 1. Localization of the epitope for Mab-1 in the three-dimensional structure of PAI-1. (A and B) Ribbon presentations of PAI-1 in the active
conformation in two different orientations. Relative to (A) the structure shown in (B) is turned approximately 180° around the y-axis and
approximately 45° around the x-axis of the coordinate system shown in the figure. Relevant secondary structural elements are marked. Please note
that T341/351 of the RCL is not visible in the structure. (C) Surface presentation of PAI-1 in the same orientation as in (B). Red residues were those
implicated in the epitope for Mab-1. Blue residues (Q57/73, Q59/75, Q61/77, K67/83, K106/125, Q109/128, R302/313, F304/315, Q305/316, T309/
319, D313/323, Q314/324, E315/325, P316/326, K325/335) were excluded from the epitope. D299/310 is indicated in yellow (see the text for details).
Nearby alanine and glycine residues (G53/69, G54/70, A62/78, A63/79 and A306/317), not testable by alanine scanning mutagenesis, are indicated
in cyan. These
SWISS PDB VIEWER
displays are based on the coordinates of Stout et al.[40].
Ó FEBS 2003 A PAI-1-protecting monoclonal antibody (Eur. J. Biochem. 270) 1673
inhibitory activity of PAI-1, its second-order rate constant
for reaction with uPA, its VN binding, or its rate of

latency transition.
The specific inhibitory activity of wt PAI-1 was
50 ± 21% (n ¼ 17) of the theoretical maximum. Most of
the mutants had a specific inhibitory activity indistinguish-
able from that of wt PAI-1. The exceptions were D313/
323A (112 ± 21%; n ¼ 17; P < 0.01) and Q58/74A-
D307/318A (15 ± 3%; n ¼ 3; P < 0.01).
Monoclonal antibodies
Mab-1 was produced and purified as described previously
[29]. Two other monoclonal antibodies against PAI-1, Mab-
2 [29,36,37] and Mab-5 [38], were produced and purified in
the same way.
Other proteins and miscellaneous materials
Bis-ANS was from Molecular Probes. S-2444 (pyro-Glu-
Gly-Arg-p-nitroanilide) was from Chromogenix (Mo
¨
lndal,
Sweden). Human urokinase-type plasminogen activator
(uPA) was from Wakamoto Pharmacautical Co. (Tokyo,
Japan). XR5118 was a kind gift from Dr Thomas Frandsen,
Finsen Laboratory, Copenhagen. The peptide TVASS,
acetylated at the N terminus and amidated at the
C terminus, was purchased from Eurogentec (Ougre
´
e,
Belgium).
ELISA
To determine the relative affinity of Mab-1 for recombinant
wt and mutant PAI-1, Mab-1 or Mab-2 was coated onto the
solid phase of microtiter wells, using an antibody concen-

tration of 2.5 lgÆmL
)1
and a buffer of 50 m
M
NaHCO
3
,
pH 9.6. After blocking with milk, dilution series of recom-
binant PAI-1, spanning a concentration range from
0.1 ngÆmL
)1
to 20 lgÆmL
)1
, were applied to the wells. The
bound PAI-1 was detected with a layer of rabbit polyclonal
anti-PAI-1 Igs, a layer of peroxidase-conjugated swine anti-
(rabbit IgG) Ig (DAKO), and a peroxidase reaction.
The 50% effective concentrations (EC
50
) for the binding
of PAI-1 to the antibodies were defined as the amount of
PAI-1 resulting in half-maximal binding.
Measurements of the effects of Mab-1 or Mab-5 and
neutralizers on the specific inhibitory activity of PAI-1
To measure the effects of antibodies and inactivators on the
specific inhibitory activity of wt and substituted PAI-1, i.e.
the fraction of inhibitor forming a stable complex with uPA,
PAI-1 was serially diluted in HBS with 0.25% gelatine,
resulting in PAI-1 concentrations between 0.01 and
20 lgÆmL

)1
in a volume of 100 lL, with or without
antibody (4 lgÆmL
)1
Mab-1 or 80 lgÆmL
)1
Mab-5). The
dilution series were then incubated for 10 min at 37 °C to
allow antibody–PAI-1 complex formation. Fifty-lL aliqu-
ots of HBS with 0.25% gelatine with or without bis-ANS or
XR5118 were added, the bis-ANS or XR5118 concentration
varying between dilution series. The mixtures were incuba-
ted for 10 min at 37 °C. Aliquots of 50 lLwith1lgÆmL
)1
uPA were added. Incubation was continued for at least
5 min, sufficient for the process of inhibition of uPA to
come to an end. The remaining uPA enzyme activity was
determined by incubation with the substrate S-2444 and
measurement of the increase in absorbance at 405 nm. The
specific inhibitory activity of PAI-1 was calculated from the
amount of PAI-1 that had to be added to inhibit 50% of
the uPA (50% inhibitory concentrations; IC
50
). The IC
50
for
bis-ANS or XR5118 neutralization of PAI-1 were deter-
mined as the neutralizer concentrations halving the PAI-1
specific inhibitory activity. The highest concentration of
XR5118 and bis-ANS used in these assays were 250 l

M
and
80 l
M
, respectively, due to solubility limits.
Measurements of the effects of Mab-1 and VN on
PAI-1 latency transition and TVASS incorporation rate
PAI-1 (20 lgÆmL
)1
)wasincubatedat37°C in HBS
supplemented with 0.25% gelatine, in the absence or
presence of Mab-1 (100 lgÆmL
)1
), VN (30 lgÆmL
)1
), and/
or the TVASS pentapeptide (250 l
M
). At regular time
intervals, samples were withdrawn for measurement of the
specific inhibitory activity of PAI-1. This was done by
making serial dilution series at 37 °C with HBS supplemen-
ted with 0.25% gelatine, resulting in PAI-1 concentrations
between 0.01 and 20 lgÆmL
)1
in a volume of 100 lL.
Aliquots of 100 lLwith0.5lgÆmL
)1
uPA were added.
After incubation for at least 2 min, the remaining uPA

enzyme activity was determined by incubation with the
chromogenic substrate S-2444 and measurement of the
increase in absorbance at 405 nm. The specific inhibitory
activity of PAI-1 was calculated from the amount of PAI-1
that had to be added to inhibit 50% of the uPA. The half-
lives of the functional activity were calculated from semi-
logarithmic plots of the specific inhibitory activity vs. time.
Statistical analysis
Data were evaluated by Student’s t-test.
Molecular graphics
SWISS PDB VIEWER
[39] was used to display the three-
dimensional structure of active PAI-1 [40].
Results
Epitope mapping
To define in detail the epitope for Mab-1, we performed
extensive alanine scanning mutagenesis around Q58/74,
already identified as being part of the epitope by Stoop
et al. [32]. Alanine-substituted and wt PAI-1s were tested
in ELISA for their binding to the antibody. The
substituted residues in variants with an EC
50
at least
twofold higher than that for wt PAI-1 were considered to
be part of the epitope. In this way, E55/71 and Q58/74 in
hC and D307/318 in the hI/s5A loop were included in the
epitope, while a number of adjacent residues were
excluded from it (Table 1, Figs 1 and 2). Combining
alanine substitutions of two of these three positions
resulted in variants with more than 20 000-fold reduced

EC
50
(Table 1). Attempts at expression of the mutant with
1674 J. S. Bødker et al. (Eur. J. Biochem. 270) Ó FEBS 2003
a triple substitution failed because of low yield. None of
the variants with substitutions in the epitope had a specific
inhibitory activity distinguishable from that of the wt. The
substitutions had no or only minor effects on binding to a
monoclonal antibody against PAI-1, Mab-2 (Table 1).
Mab-2 has an epitope of residues in hF and its flanking
sequences [37].
Mab-1 protection of PAI-1 against bis-ANS and XR5118
The IC
50
values for bis-ANS and XR5118 inactivation of wt
PAI-1 were 0.62 ± 0.06 (n ¼ 6) and 10.1 ± 3.0 (n ¼ 11)
l
M
, respectively, in agreement with previous reports
[11,24,28]. We now found that the IC
50
values for bis-
ANS and XR5118 inactivation of the Mab-1–PAI-1
complex were higher than 80 l
M
(n ¼ 3) and higher than
250 l
M
(n ¼ 12), respectively. Thus, Mab-1 protects wt
PAI-1 against these neutralizers. Similar observations were

done with two other neutralizers, 1-anilinonaphtalene-8-
sulfonic acid and 1-dodecyl sulphuric acid (data not shown).
In contrast, the monoclonal antibody against PAI-1 Mab-5,
having an epitope not overlapping that of Mab-1 [38], did
not protect PAI-1 against XR5118 and bis-ANS. The IC
50
values for bis-ANS and XR5118 inactivation of the Mab-5-
PAI-1 complex were 0.57 ± 0.02 (n ¼ 3) and 9.8 ± 2.23
(n ¼ 6), respectively, not significantly different from the
values without antibody (P < 0.01). In the absence of
inactivators, neither antibody affected the specific inhibitory
activity of PAI-1.
To analyse the effect of the amino acid substitutions in
and around the epitope of Mab-1 on the ability to protect
against XR5118, we measured the specific inhibitory activity
of each variant with alanine substitutions in the absence and
presence of Mab-1, and in the presence of XR5118 at
concentrations between 0 and 80 l
M
. In the absence of
XR5118, Mab-1 did not affect the specific inhibitory activity
of any of the variants, and all variants had IC
50
values for
inactivation by XR5118 indistinguishable from that of wt
(data not shown). Whereas wt PAI-1 was totally resistant to
80 l
M
XR5118inthepresenceofMab-1,someofthe
mutants were only partially or not at all protected against

XR5118 by Mab-1 (Fig. 3). As expected, Mab-1 gave little
or no protection to the variants with substitutions in the
epitope, i.e., E55/71A, Q58/74A, and D307/318A, and the
double mutants E55/71A-Q58/74A, E55/71A-D307/318A
and Q58/74A-D307/318A. In addition, D299/310A was
incompletely protected by Mab-1.
Mab-1 and PAI-1 latency transition and PAI-1
inactivation by an insertion peptide
In the presence of Mab-1, the half-life for latency transition
of PAI-1 was increased by a factor of  1.5. The half-lives in
the presence of Mab-1 and in the presence of VN were
indistinguishable. However, with the variant K325A, the
effects of VN and Mab-1 were different. This variant has a
twofold increased half-life as compared to wt, and the half-
life is not increased, but decreased by VN. We found now
that Mab-1 did not affect the latency transition rate of this
variant (Table 2 and Fig. 4). Thus, Mab-1 and VN affect
the latency transition rate by different mechanisms.
Table 1. Effect of alanine substitutions on the affinity of PAI-1 to Mab-1. The EC
50
values for binding of each variant to Mab-1 or Mab-2 were
determined in parallel with the EC
50
value for wt and expressed as a fraction of that. The means and standard deviations of triple determinations are
indicated. Besides the results shown in the table, the following variants were tested, but found to be indistinguishable from wt with respect
to the affinity to Mab-1: Q57/73A, Q59/75A, Q61/77A, K67/83A, K106/125A, Q109/128A, D299/310A, R302/313A, F304/315A, Q305/316A,
T309/319A, D313/323A, Q314/324A, E315/325A, P316/326A, K325/335A.
Substitution(s)
Secondary structural
element

Amino acid in
murine PAI-1
Mab-1
EC
50variant
/EC
50wt
Mab-2
EC
50variant
/EC
50wt
E55/71A hC K 22.7 ± 7.3
a
1.1 ± 0.1
Q58/74A hC R 6.6 ± 1.5
a
1.3 ± 0.2
D307/318A hI/s5A loop D 3.6 ± 0.7
a
1.8 ± 0.5
E55/71A-Q58/74A hC K and R >20 000
a
1.6 ± 0.3
E55/71A-D307/318A hC and hI/s5A loop K and D >20 000
a
3.6 ± 1.1
Q58/74A-D307/318A hC and hI/s5A loop R and D >20 000
a
2.6 ± 0.5

a
a
Significantly different from 1 (P < 0.025).
Fig. 2. Localization of the amino acids in the epitope for Mab-1. The
structure shown is a ribbon representation of the coordinates of Stout
et al. [40]. The side chains of the amino acids in the epitope and D299/
310 are displayed as sticks and CPK-coloured. The colours of the
secondary structure elements are identical to those in Fig. 1 (see text
for further details).
Ó FEBS 2003 A PAI-1-protecting monoclonal antibody (Eur. J. Biochem. 270) 1675
Two molecules of the pentapeptide TVASS is able to
insert between s3A and s5A in active PAI-1, mimicking the
RCL of relaxed forms of PAI-1. The (TVASS)
2
–PAI-1
complex displays substrate behaviour, presumably due to a
reduced rate of RCL insertion during the reaction with a
target proteinase [41]. We measured the effect of Mab-1 on
the rate of incorporation of TVASS into PAI-1 by
measuring the specific inhibitory activity of PAI-1 after
incubation with TVASS at 37 °C for different time periods
in the absence or presence of Mab-1. However, Mab-1 did
not affect the rate of TVASS-induced inactivation of PAI-1,
the half-life for inactivation by 250 l
M
TVASS being
10.3 ± 2.7 min (n ¼ 3) in the absence of Mab-1 and
12.9 ± 0.9 min (n ¼ 3) in the presence of Mab-1.
Discussion
To the best of our knowledge, Mab-1 is the only mono-

clonal antibody known to stabilize PAI-1 in an inhibitory
active form. We previously reported that Mab-1 stabilizes
PAI-1 against cold-induced substrate behaviour in buffers
with nonionic detergents [30]. We report here that Mab-1
delays PAI-1 latency transition and protects PAI-1 against
bis-ANS- and XR5118-induced inactivation.
As monitored by proteolytic susceptibility, the protection
by Mab-1 against cold-induced substrate behaviour in
detergent-containing buffers is associated with conforma-
tional changes of the RCL, the sequence Q321/331-K325/
335 in s5A and of the flexible joint region [30,31]. Bis-ANS
induces substrate behaviour and polymerization, and
XR5118 induces conversion to an inert monomeric form
[11]. Taken together, these observations show that Mab-1
stabilizes the inhibitory activity of PAI-1 against inactiva-
tion by affecting the conformation of the flexible joint
region, the central sequence of s5A, and/or the RCL.
Stoop et al. [32] initially reported that Q58/74 is import-
ant for binding of PAI-1 to Mab-1. We have demonstrated
here that the epitope also includes E55/71 and D307/318A.
In agreement with expectancies for a murine antibody
against a human protein, two of the residues in the epitope
are different in humans and mice (Table 1). The epitope
spans residues in both hC and the loop connecting hI and
s5A. It is thus obvious that Mab-1 affects interactions of
residues of PAI-1 which are localized distantly from its
epitope. We used a combination of site-directed mutagenesis
and Mab-1 protection of PAI-1 against XR5118-induced
inactivation to obtain information about how the conform-
ational change initiated by the binding of Mab-1 spreads

through the molecule. Generally, observation of a substi-
tution having a different effect on XR5118 inactivation of
PAI-1 in the absence and presence of Mab-1 shows that the
Fig. 3. Effect of XR5118 on the specific inhibitory activities of wt PAI-1
and PAI-1 variants in the absence and the presence of Mab-1. The
specific inhibitory activities of PAI-1 in the absence and presence of
Mab-1 were measured in the presence of the indicated concentrations
of XR5118 and expressed relative to the specific inhibitory activity in
the absence of XR5118. Means and standard deviations are indicated.
The four mutants shown are significantly different from wt with respect
to residual inhibitory activity in the presence of Mab-1 and 80 l
M
XR5118 (P < 0.01). Besides the variants shown in the figure, we
tested the following variants and found that they did not differ from wt
with respect to the response of the specific inhibitory activity to Mab-1:
Q57/73A, Q59/75A, Q61/77A, K67/83A, K106/125A, Q109/128A,
R302/313A, F304/315A, Q305/316A, T309/319A, D313/323A, Q314/
324A, E315/325A, P316/326A, K325/335A.
1676 J. S. Bødker et al. (Eur. J. Biochem. 270) Ó FEBS 2003
corresponding amino acid side chain is in different sur-
roundings in the absence and presence of Mab-1. Accord-
ingly, all the variants with substitutions in the Mab-1
epitope were susceptible to XR5118 in the presence of
Mab-1. In addition, substitution of D299/310, localized in
the hI-s5A loop (Fig. 2), but outside the epitope, also
resulted in a reduced ability of Mab-1 to protect PAI-1
against XR5118. A Mab-1-induced reorientation of D299/
310 may therefore be important for the transmission of a
Mab-1-induced signal from the epitope to b-sheet A, the
RCL, and the flexible joint region.

It is interesting to note that whereas Mab-1 delayed the
rate of latency transition, it did not measurably affect the
rate of incorporation of TVASS into b-sheet A. This
observation is in agreement with the notion that the rate-
limiting step during latency transition is not insertion of the
RCL into b-sheet A, but rather passage of the RCL through
the gate region [42]. Anyway, the mechanism by which
Mab-1 delays latency transition is different from that of
VN, as we demonstrate here that the two have different
effects on PAI-1 K325/335A.
Conclusively, on the basis of the reported observations,
we propose that the binding of Mab-1 to PAI-1 results in a
conformational change of hC and the hI-s5A loop which
spreads to the flexible joint region, the central portion of
s5A, and the RCL, and thus affects the functional properties
of PAI-1.
PAI-1 is a potential target for antithrombotic and
anticancer therapy (for a review see [20]). A variety of
model systems is available for studying the effects of PAI-1
inactivators on thrombi and tumours (for reviews see
[43–45]). By stabilizing PAI-1 against inactivation, Mab-1
may be used as a valuable reagent for controlling specificity
for PAI-1 inactivators in such model systems.
References
1. Ye, S., Cech, A.L., Belmares, R., Bergstro
¨
m, R.C., Tong, Y.,
Corey, D.R., Kanost, M.R. & Goldsmith, E.J. (2001) The struc-
ture of a Michaelis serpin-protease complex. Nature Struct. Biol.
8, 979–983.

2. Wilczynska, M., Fa, M., Ohlsson, P.I. & Ny, T. (1995) The
inhibition mechanism of serpins. Evidence that the mobile reactive
center loop is cleaved in the native protease-inhibitor complex.
J. Biol. Chem. 270, 29652–29655.
3. Lawrence, D.A., Ginsburg, D., Day, D.E., Berkenpas, M.B.,
Verhamme, I.M., Kvassman, J.O. & Shore, J.D. (1995) Serpin-
protease complexes are trapped as stable acyl-enzyme inter-
mediates. J. Biol. Chem. 270, 25309–25312.
4. Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen,
M.S., Guldberg, R.E. & Petersen, T.E. (1998) An ester bond
linking a fragment of a serine proteinase to its serpin inhibitor.
Biochemistry 37, 6375–6379.
5. Shore, J.D., Day, D.E., Francis-Chmura, A.M., Verhamme, I.,
Kvassman,J.,Lawrence,D.A.&Ginsburg,D.(1995)Afluor-
escent probe study of plasminogen activator inhibitor-1. Evidence
for reactive center loop insertion and its role in the inhibitory
mechanism. J. Biol. Chem. 270, 5395–5398.
6. Stratikos, E. & Gettins, P.G. (1999) Formation of the covalent
serpin-proteinase complex involves translocation of the proteinase
by more than 70 A
˚
and full insertion of the reactive center loop
into b-sheet A. Proc. Natl Acad. Sci. USA 96, 4808–4813.
7. Fa, M., Bergstro
¨
m, F., Hagglo
¨
f, P., Wilczynska, M., Johansson,
L.B. & Ny, T. (2000) The structure of a serpin-protease complex
revealed by intramolecular distance measurements using donor-

donor energy migration and mapping of interaction sites. Struc-
ture Fold. Des. 8, 397–405.
8. Huntington, J.A., Read, R.J. & Carrell, R.W. (2000) Structure of a
serpin-protease complex shows inhibition by deformation. Nature
407, 923–926.
Fig. 4. Effect of Mab-1 on the rate of latency transition of PAI-1 wt and
K325/335A. PAI-1 wt and PAI-1 K523/335 A were incubated at 37 °C
with or without Mab-1 or VN for various times, followed by meas-
urements of the remaining specific inhibitory activity by titration
against uPA. The activities are given relative to the initial activity. The
graphs show the results of representative experiments. Data from all
determinations are shown in Table 2.
Table 2. Effect of Mab-1 on the rate of latency transition of PAI-1.
PAI-1 alone, with Mab-1, or with VN, was incubated at 37 °C;the
PAI-1 concentration was 20 lgÆmL
)1
, the Mab-1 concentration was
100 lgÆmL
)1
, and the VN concentration was 30 lgÆmL
)1
.Aftervari-
ous incubation times samples were taken for determination of the
specific inhibitory activity of PAI-1. The specific inhibitory activities
measured were plotted semilogarithmically vs. incubation time, and
the half-lives were calculated from the slopes of the lines by linear
regression analysis.
PAI-1
variant
Incubation

condition
Half-life
[mean ± SD (n)]
wt No additions 58 ± 3 (4)
+Mab-1 81 ± 7 (3)
a
+VN 81 ± 14 (3)
a
K325/335A No additions 116 ± 6 (3)
b
+Mab-1 126 ± 4 (3)
b
+VN 67 ± 1 (3)
a
a
Significantly different from the corresponding value without
additions (P ¼ 0.01).
b
Significantly different from the corres-
ponding value for wt (P < 0.01).
Ó FEBS 2003 A PAI-1-protecting monoclonal antibody (Eur. J. Biochem. 270) 1677
9. Plotnick, M.I., Mayne, L., Schechter, N.M. & Rubin, H. (1996)
Distortion of the active site of chymotrypsin complexed with a
serpin. Biochemistry 35, 7586–7590.
10. Stavridi, E.S., O’Malley, K., Lukacs, C.M., Moore, W.T., Lam-
bris, J.D., Christianson, D.W., Rubin, H. & Cooperman, B.S.
(1996) Structural change in a-chymotrypsin induced by complex-
ation with a1-antichymotrypsin as seen by enhanced sensitivity to
proteolysis. Biochemistry 35, 10608–10615.
11. Egelund, R., Petersen, T.E. & Andreasen, P.A. (2001) A serpin-

induced extensive proteolytic susceptibility of urokinase-type
plasminogen activator implicates distortion of the proteinase
substrate-binding pocket and oxyanion hole in the serpin
inhibitory mechanism. Eur. J. Biochem. 268, 673–685.
12. Fredenburgh, J.C., Stafford, A.R. & Weitz, J.I. (2001) Con-
formational changes in thrombin when complexed by serpins.
J. Biol. Chem. 276, 44828–44834.
13. Peterson, F.C. & Gettins, P.G. (2001) Insight into the mechanism
of serpin-proteinase inhibition from 2D [1H)15N] NMR studies
of the 69 kDa a1-proteinase inhibitor Pittsburgh-trypsin covalent
complex. Biochemistry 40, 6284–6292.
14. Tew, D.J. & Bottomley, S.P. (2001) Intrinsic fluorescence changes
and rapid kinetics of proteinase deformation during serpin
inhibition. FEBS Lett. 494, 30–33.
15. Plotnick, M.I., Samakur, M., Wang, Z.M., Liu, X., Rubin, H.,
Schechter, N.M. & Selwood, T. (2002) Heterogeneity in serpin-
protease complexes as demonstrated by differences in the
mechanism of complex breakdown. Biochemistry 41, 334–342.
16. Ludeman, J.P., Whisstock, J.C., Hopkins, P.C., Le Bonniec, B.F.
& Bottomley, S.P. (2001) Structure of a serpin-enzyme complex
probed by cysteine substitutions and fluorescence spectroscopy.
Biophys. J. 80, 491–497.
17. Gils, A. & Declerck, P.J. (1998) Structure-function relationships in
serpins: current concepts and controversies. Thromb. Haemost. 80,
531–541.
18. Irving, J.A., Pike, R.N., Lesk, A.M. & Whisstock, J.C. (2000)
Phylogeny of the serpin superfamily: implications of patterns of
amino acid conservation for structure and function. Genome Res.
10, 1845–1864.
19. Ye, S. & Goldsmith, E.J. (2001) Serpins and other covalent pro-

tease inhibitors. Curr. Opin. Struct. Biol. 11, 740–745.
20. Wind, T., Hansen, M., Jensen, J.K. & Andreasen, P.A. (2002) The
molecular basis for anti-proteolytic and non-proteolytic functions
of plasminogen activator inhibitor type-1: roles of the reactive
centre loop, the shutter region, the flexible joint region and the
small serpin fragment. Biol. Chem. 383, 21–36.
21. Mottonen, J., Strand, A., Symersky, J., Sweet, R.M., Danley,
D.E.,Geoghegan,K.F.,Gerard,R.D.&Goldsmith,E.J.(1992)
Structural basis of latency in plasminogen activator inhibitor-1.
Nature 355, 270–273.
22. Carrell, R.W., Huntington, J.A., Mushunje, A. & Zhou, A. (2001)
The conformational basis of thrombosis. Thromb. Haemost. 86,
14–22.
23. Lawrence, D.A., Berkenpas, M.B., Palaniappan, S. & Ginsburg,
D. (1994) Localization of vitronectin binding domain in plasmi-
nogen activator inhibitor-1. J. Biol. Chem. 269, 15223–15228.
24. Jensen, J.K., Wind, T. & Andreasen, P.A. (2002) The vitronectin
binding area of plasminogen activator inhibitor-1, mapped by
mutagenesis and protection against an inactivating organochem-
ical ligand. FEBS Lett. 521, 91–94.
25. Bjo
¨
rquist, P., Ehnebom, J., Inghardt, T., Hansson, L., Lindberg,
M., Linschoten, M., Stro
¨
mqvist,M.&Deinum,J.(1998)Identi-
fication of the binding site for a low-molecular-weight inhibitor of
plasminogen activator inhibitor type 1 by site-directed mutagen-
esis. Biochemistry 37, 1227–1234.
26. Friederich, P.W., Levi, M., Biemond, B.J., Charlton, P.,

Templeton, D., van Zonneveld, A.J., Bevan, P., Pannekoek, H. &
ten Cate, J.W. (1997) Novel low-molecular-weight inhibitor of
PAI-1 (XR5118) promotes endogenous fibrinolysis and reduces
post-thrombolysis thrombus growth in rabbits. Circulation 96,
916–921.
27. Egelund, R., Einholm, A.P., Pedersen, K.E., Nielsen, R.W.,
Christensen, A., Deinum, J. & Andreasen, P.A. (2001) A
regulatory hydrophobic area in the flexible joint region of plas-
minogen activator inhibitor-1, defined with fluorescent activity-
neutralizing ligands. Ligand-induced serpin polymerization.
J. Biol. Chem. 276, 13077–13086.
28. Jensen, S., Kirkegaard, T., Pedersen, K.E., Busse, M., Preissner,
K.T., Rodenburg, K.W. & Andreasen, P.A. (2002) The role of
b-strand 5A of plasminogen activator inhibitor-1 in regulation
of its latency transition and inhibitory activity by vitronectin.
Biochim. Biophys. Acta 1597, 301–310.
29. Nielsen, L.S., Andreasen, P.A., Grøndahl-Hansen, J., Huang,
J.Y., Kristensen, P. & Danø, K. (1986) Monoclonal antibodies to
human 54,000 molecular weight plasminogen activator inhibitor
from fibrosarcoma cells – inhibitor neutralization and one-step
affinity purification. Thromb. Haemost. 55, 206–212.
30. Kjøller, L., Martensen, P.M., Sottrup-Jensen, L., Justesen, J.,
Rodenburg, K.W. & Andreasen, P.A. (1996) Conformational
changes of the reactive-centre loop and b-strand 5A accompany
temperature-dependent inhibitor-substrate transition of plasmi-
nogen-activator inhibitor 1. Eur. J. Biochem. 241, 38–46.
31. Kirkegaard, T., Jensen, S., Schousboe, S.L., Petersen, H.H.,
Egelund, R., Andreasen, P.A. & Rodenburg, K.W. (1999)
Engineering of conformations of plasminogen activator inhibitor-
1. A crucial role of b-strand 5A residues in the transition of

active form to latent and substrate forms. Eur. J. Biochem. 263,
577–586.
32. Stoop, A.A., Jespers, L., Lasters, I., Eldering, E. & Pannekoek, H.
(2000) High-density mutagenesis by combined DNA shuffling and
phage display to assign essential amino acid residues in protein–
protein interactions: application to study structure-function of
plasminogen activation inhibitor 1 (PAI-I). J. Mol. Biol. 301,
1135–1147.
33. Andreasen,P.A.,Riccio,A.,Welinder,K.G.,Douglas,R.,Sar-
torio, R., Nielsen, L.S., Oppenheimer, C., Blasi, F. & Dano, K.
(1986) Plasminogen activator inhibitor type-1: reactive center and
amino-terminal heterogeneity determined by protein and cDNA
sequencing. FEBS Lett. 209, 213–218.
34. Huber, R. & Carrell, R.W. (1989) Implications of the three-
dimensional structure of a1-antitrypsin for structure and function
of serpins. Biochemistry 28, 8951–8966.
35. Christensen, J.H., Hansen, P.K., Lillelund, O. & Thøgersen, H.C.
(1991) Sequence-specific binding of the N-terminal three-finger
fragment of Xenopus transcription factor IIIA to the internal
control region of a 5S RNA gene. FEBS Lett. 281, 181–184.
36. Schousboe, S.L., Egelund, R., Kirkegaard, T., Preissner, K.T.,
Rodenburg, K.W. & Andreasen, P.A. (2000) Vitronectin and
substitution of a b-strand 5A lysine residue potentiate activity-
neutralization of PA inhibitor-1 by monoclonal antibodies against
a-helix F. Thromb. Haemost. 83, 742–751.
37. Wind, T., Jensen, M.A. & Andreasen, P.A. (2001) Epitope map-
ping for four monoclonal antibodies against human plasminogen
activator inhibitor type-1: implications for antibody-mediated
PAI-1-neutralization and vitronectin-binding. Eur. J. Biochem.
268, 1095–1106.

38. Munch, M., Heegaard, C., Jensen, P.H. & Andreasen, P.A. (1991)
Type-1 inhibitor of plasminogen activators. Distinction between
latent, activated and reactive centre-cleaved forms with thermal
stability and monoclonal antibodies. FEBS Lett. 295, 102–106.
39. Guex, N. & Peitsch, M.C. (1997) SWISS-MODEL and the Swiss-
PdbViewer: an environment for comparative protein modeling.
Electrophoresis 18, 2714–2723.
1678 J. S. Bødker et al. (Eur. J. Biochem. 270) Ó FEBS 2003
40. Stout, T.J., Graham, H., Buckley, D.I. & Matthews, D.J.
(2000) Structures of active and latent PAI-1: a possible
stabilizing role for chloride ions. Biochemistry 39, 8460–
8469.
41. Xue, Y., Bjo
¨
rquist, P., Inghardt, T., Linschoten, M., Musil, D.,
Sjo
¨
lin, L. & Deinum, J. (1998) Interfering with the inhibitory
mechanism of serpins: crystal structure of a complex formed
between cleaved plasminogen activator inhibitor type 1 and a
reactive-centre loop peptide. Structure 6, 627–636.
42. Kruger, P., Verheyden, S., Declerck, P.J. & Engelborghs, Y.
(2001) Extending the capabilities of targeted molecular dynamics:
simulation of a large conformational transition in plasminogen
activator inhibitor 1. Protein Sci. 10, 798–808.
43. Andreasen,P.A.,Kjøller,L.,Christensen,L.&Duffy,M.J.(1997)
The urokinase-type plasminogen activator system in cancer
metastasis: a review. Int. J. Cancer 72, 1–22.
44. Andreasen, P.A., Egelund, R. & Petersen, H.H. (2000) The plas-
minogen activation system in tumor growth, invasion, and

metastasis. Cell Mol. Life Sci. 57, 25–40.
45. Huber, K., Christ, G., Wojta, J. & Gulba, D. (2001) Plasminogen
activator inhibitor type-1 in cardiovascular disease. Status report
2001. Thromb. Res. 103 (Suppl. 1), S7–S19.
Ó FEBS 2003 A PAI-1-protecting monoclonal antibody (Eur. J. Biochem. 270) 1679