Tài liệu Báo cáo khoa học: Oxidized elafin and trappin poorly inhibit the elastolytic activity of neutrophil elastase and proteinase 3 pdf

Oxidized elafin and trappin poorly inhibit the elastolytic
activity of neutrophil elastase and proteinase 3
Shila M. Nobar
1
, Marie-Louise Zani
2
, Christian Boudier
1
, Thierry Moreau
2
and Joseph G. Bieth
1
1 Laboratoire d’Enzymologie, INSERM U392, Universite
´
Louis Pasteur de Strasbourg, Illkirch, France
2 INSERM U618, Universite
´
Franc¸ois Rabelais, Tours, France
Many amino acid residues of proteins are susceptible to
oxidation by reactive oxygen species. Methionine, the
most sensitive of amino acids to oxidation, is readily
transformed into a mixture of the S- and R-epimers of
methionine sulfoxide. The latter may be recycled by
methionine sulfoxide reductases in the presence of thio-
redoxin, which itself may be regenerated by thioredoxin
reductase in an NADPH-dependent reaction. Excessive
methionine sulfoxide production and ⁄ or a defect in its
recycling is believed to be involved in age-related
diseases and in shortening of the maximum life span [1].
Oxidative processes also take place in lung infection
and inflammation, where they are used, in conjunction

with proteolytic enzymes, to kill bacteria and destroy
foreign substances in the phagolysosome of polymor-
phonuclear neutrophils. The membrane of these phago-
cytes contains an NADPH oxidase, which transforms
molecular oxygen into the short-lived superoxide
anion. Superoxide dismutase transforms the latter into
H
2
O
2
, an oxidant that further yields hypochloride in
the presence of neutrophil myeloperoxidase. Aliphatic
amines transform hypochloride into chloramines,
which are potent and long-lived oxidants [2].
In inflammatory lung diseases, such as chronic bron-
chitis, emphysema or cystic fibrosis, excessive recruit-
ment, activation or lysis of neutrophils results in
the extracellular release of neutrophil elastase (NE;
EC 3.4.21.37), proteinase 3 (Pr3; EC 3.4.21.76) and
Keywords
elafin; elastase; enzyme kinetics; oxidation;
proteinase 3
Correspondence
J. G. Bieth, INSERM U 392, Faculte
´
de
Pharmacie, 74 route du Rhin,
67400 Illkirch, France
Fax: +33 3 90 24 43 08
Tel: +33 3 90 24 41 82

E-mail:
(Received 20 May 2005, revised 24 August
2005, accepted 22 September 2005)
doi:10.1111/j.1742-4658.2005.04988.x
Neutrophil proteinase-mediated lung tissue destruction is prevented by
inhibitors, including elafin and its precursor, trappin. We wanted to estab-
lish whether neutrophil-derived oxidants might impair the inhibitory func-
tion of these molecules. Myeloperoxidase ⁄ H
2
O
2
and N-chlorosuccinimide
oxidation of the inhibitors was checked by mass spectrometry and enzy-
matic methods. Oxidation significantly lowers the affinities of the two
inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This
decrease in affinity is essentially caused by an increase in the rate of inhibi-
tory complex dissociation. Oxidized elafin and trappin have, however, rea-
sonable affinities for NE (K
i
¼ 4.0–9.2 · 10
)9
m) and for Pr3 (K
i
¼ 2.5–
5.0 · 10
)8
m). These affinities are theoretically sufficient to allow the oxi-
dized inhibitors to form tight binding complexes with NE and Pr3 in lung
secretions where their physiological concentrations are in the micromolar
range. Yet, they are unable to efficiently inhibit the elastolytic activity of

the two enzymes. At their physiological concentration, fully oxidized elafin
and trappin do not inhibit more than 30% of an equimolar concentration
of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin
strongly impairs their activity. Inhibitor-based therapy of inflammatory
lung diseases must be carried out using oxidation-resistant variants of these
molecules.
Abbreviations
Lys-(pico), lysyl-(2-picolinoyl); MeOSuc, methoxysuccinyl; NE, human neutrophil elastase; pNA, p-nitroanilide; Pr3, human neutrophil
proteinase 3; RBB–elastin, remazol-Brilliant Blue–elastin.
FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS 5883
cathepsin G, three neutral serine proteinases that have
been shown in vitro to cleave lung extracellular matrix
proteins, including elastin, collagens, fibronectin and
laminin. These enzymes are thought to be responsible
for lung tissue destruction [2,3].
Nature has designed potent protein proteinase inhib-
itors to prevent local proteolysis caused by accidental
neutrophil proteinase release during normal breathing,
where inhalation of micro-organisms and air pollutants
always takes place. These proteins include a
1
-protein-
ase inhibitor (also called a
1
-antitrypsin; a 53-kDa pro-
tein that inhibits the above three enzymes) [3];
a
1
-antichymotrypsin (a 68-kDa protein that specifically
inhibits cathepsin G) [4]; mucus proteinase inhibitor

(also called secretory leukoprotease inhibitor, or SLPI;
an 11.7-kDa protein that inhibits NE [5] and cathepsin
G, but not Pr3 [3]); and elafin and its precursor trap-
pin-2 (also called pre-elafin and referred to as trappin
throughout this article; that inhibit NE and Pr3 [6],
but not cathepsin G [3]). The two former proteins are
mainly synthesized in the liver and reach the lung via
the blood circulation. They are irreversible inhibitors
that belong to the serpin family. Their interaction with
proteinases is characterized by a single constant – the
association rate constant (E þ I À!
k
ass
EI) [7]. The two lat-
ter molecules are synthesized in the lung and belong to
the canonical type of inhibitors that interact reversibly
with their target enzymes, the reaction being described
by an association and a dissociation rate constant
and E þ I Ð
k
ass
k
diss
EI an equilibrium dissociation constant
K
i
¼ k
diss
⁄ k
ass

[5,8].
Trappin is a 9.9-kDa protein formed of two proteo-
lytically cleavable domains. Four disulfide bridges sta-
bilize its 6-kDa C-terminal inhibitory domain, named
elafin in this article [9,10]. Its N-terminal domain, the
so-called cementoin domain, contains four repeats,
with a Gly–Gln–Asp–Pro–Val–Lys consensus sequence
homologous to a putative transglutaminase substrate
motif. The trappin molecule may therefore be cova-
lently attached to other proteins [11]. These inhibitors
are also antimicrobial [12,13] and thus participate in
innate immunity [14].
We have recently used the Pichia pastoris expression
system to prepare elafin and trappin in high yields.
The two full-length recombinant inhibitors were found
to be virtually indistinguishable in their kinetic con-
stants for the inhibition of NE and Pr3: both were
fast-acting inhibitors with k
ass
¼ 2–4 · 10
6
m
)1
Æs
)1
and
formed very stable inhibitory complexes with k
diss
and
K

i
in the 10
)4
Æs
)1
and 10
)10
m range, respectively [15].
In inflammatory lung diseases, activated or lysed
neutrophils do not only release proteinases but also
the aforementioned oxidants. The present article
reports the kinetic consequences of inhibitor elafin and
trappin oxidation on their interaction with NE and
Pr3. It also examines the effect of insoluble elastin on
the inhibitory properties of the native and oxidized
inhibitors.
Results
Oxidation decreases the affinity of elafin and
trappin for NE and Pr3
We oxidized elafin and trappin using either N-chloro-
succinimide, a classical reagent for surface-exposed
methionine residues [16] or with the myelopero xidase ⁄
H
2
O
2
⁄ halide system, the neutrophil’s oxidation device
[17]. Figure 1 shows the effect of increasing concen-
trations of native and oxidized elafin and trappin on
the activity of a constant concentration of NE and

Fig. 1. Inhibition of neutrophil elastase (NE) and proteinase 3 (Pr3)
by native and N-chlorosuccinimide-oxidized elafin (A) and trappin
(B). Increasing concentrations of inhibitor were added to constant
concentrations of enzyme, and the residual enzymatic activities
were measured using appropriate substrates. (h), Native inhibitors
+NEorPr3;(s), (D), oxidized inhibitors + NE or Pr3, respectively.
Inhibition of elastase and proteinase 3 by elafin S. M. Nobar et al.
5884 FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS
Pr3. Straight inhibition curves were obtained with the
native inhibitors, in agreement with the low values of
K
i
[15], as compared with the enzyme concentrations
used in the present assays [18]. In contrast, the curves
describing the inhibition of NE by the oxidized inhibi-
tors were concave, indicating a significant decrease in
affinity [18]. The inhibition of Pr3 was even more dra-
matically affected: an equimolar solution of enzyme +
oxidized inhibitor yielded only about 50% inhibition.
To express the oxidation effect in a quantitative
manner, we measured the equilibrium dissociation con-
stant, K
i
, for the complexes formed of oxidized elafin
or trappin and NE or Pr3. Oxidation was carried out
with either N-chlorosuccinimide or myeloperoxidase.
The K
i
values were determined from inhibition curves,
such as those shown in Fig. 1. These curves were ana-

lyzed using Eqn (1):
a ¼ 1 À
ð½E
0
þ½I
0
þ KÞÀ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½E
0
þ½I
0
þ KÞ
2
À 4½E
0
½I
0
q
2½E
0
ð1Þ
where a is the relative enzyme activity (rate in the
presence of inhibitor ⁄ rate in its absence), [E]
0
and [I]
0
are the total enzyme and inhibitor concentrations,
respectively, and K ¼ K
i

if the substrate (S) does not
dissociate EI during the 20–60 s assay of enzymatic
activity or K ¼ K
i
(1+[S]
0
⁄ K
m
) if there is partial disso-
ciation of EI by S so that E, I, S are in equilibrium
with ES and EI. Substrate-induced dissociation experi-
ments (see below) showed that dissociation of the
oxidized inhibitor–NE complex was slow enough to
be insignificant during the 20–60 s time period used to
measure the activities of the inhibitory mixtures.
Therefore, the K of Eqn (1) is substrate-independent
and equals K
i
. In contrast, dissociation of the oxidized
inhibitor–Pr3 complex was very fast, so that E, I, S
and their complexes were in equilibrium following the
time required to mix the reagents. Hence, K is sub-
strate-dependent and equals K
i
(1 + [S]
0
⁄ K
m
). As
shown in Table 1, oxidation of elafin and trappin sig-

nificantly increases the K
i
(decreases the affinity) for
its complexes with NE and Pr3. Oxidation by N-chloro-
succinimide or myeloperoxidase yields inhibitors
whose K
i
values are not significantly different from
each other.
Oxidized elafin and trappin form unstable
complexes with NE and Pr3
Is the above-observed increase in K
i
caused by an
increase in the dissociation rate constant, k
diss
,a
decrease in the association rate constant, k
ass
,oran
effect on both parameters (K
i
¼ k
diss
⁄ k
ass
)? To answer
this question, we measured k
diss
by extensively diluting

equimolar enzyme–inhibitor solutions into highly con-
centrated substrate solutions and following the hydro-
lysis of substrate as a function of time. The complexes
formed of NE and native or N-chlorosuccinimide-oxi-
dized elafin and trappin gave progress curves that were
initially concave, indicating continuous release of free
enzyme, that is, complex dissociation. After a time, the
curves became linear, indicating that the enzyme–
inhibitor–substrate system had reached its steady state
(Fig. 2A). Comparison of the time required to reach
this steady state, and of the steady-state rates, clearly
shows that the NE-oxidized inhibitor complexes disso-
ciate much faster than the NE-native inhibitor ones.
Table 1. Kinetic constants describing the inhibition of neutrophil elastase (NE) and proteinase 3 (Pr3) by oxidized elafin and trappin. The data
for the native inhibors are from Zani et al. [15]. The k
diss
and K
i
values are experimental, whereas the k
ass
values are calculated. MPO,
myeloperoxidase ⁄ H
2
O
2
⁄ Cl
–
; NCS, N-chlorosuccinimide; ND, not determined.
Enzyme Inhibitor Oxidant K
i

(M) k
ass
(M
)1
Æs
)1
) k
diss
(s
)1
)
NE Elafin None 8.0 ± 0.5 · 10
)11
3.7 ± 0.1 · 10
6
3.2 ± 0.1 · 10
)4
NCS 5.7 ± 0.6 · 10
)9
1.1 ± 0.3 · 10
6
6.3 ± 0.6 · 10
)3
MPO 4.0 ± 0.6 · 10
)9
ND ND
NE Trappin None 3.0 ± 1.0 · 10
)11
3.6 ± 0.5 · 10
6

1.1 ± 0.2 · 10
)4
NCS 9.2 ± 2.7 · 10
)9
1.0 ± 0.3 · 10
6
9.0 ± 3.1 · 10
)3
MPO 5.8 ± 0.9 · 10
)9
ND ND
Pr3 Elafin None 1.2 ± 0.1 · 10
)10
3.3 ± 0.03 · 10
6
4.0 ± 0.3 · 10
)4
NCS 2.9 ± 0.2 · 10
)8
ND ‡ 0.1
a
MPO 2.5 ± 0.2 · 10
)8
ND ND
Pr3 Trappin None 1.8 ± 0.6 · 10
)10
2.0 ± 0.1 · 10
6
3.7 ± 1.1 · 10
)4

NCS 5.0 ± 2.0 · 10
)8
ND ‡ 0.1
a
MPO 3.5 ± 0.5 · 10
)8
ND ND
a
Calculated assuming that dissociation is terminated in 30 s or less, which corresponds to a t
½
£ 6s.
S. M. Nobar et al. Inhibition of elastase and proteinase 3 by elafin
FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS 5885
Quantitative calculation of k
diss
confirms this
(Table 1). The complexes formed of Pr3 and oxidized
elafin and trappin were found to dissociate within the
mixing time because no presteady state was visible
(Fig. 2, curves 1 and 2). Hence, k
diss
could not be cal-
culated for these systems but is estimated to be greater
than 0.1 s
)1
(Table 1 legend). Thus, the oxidation of
elafin and trappin leads to a > 250-fold increase of
k
diss
of their complexes with Pr3. We conclude that the

oxidation of elafin and trappin renders the inhibitors
unable to form stable complexes with NE and Pr3.
Similar results were observed with trappin. Calculation
of k
ass
for the NE-oxidized elafin and trappin com-
plexes using the measured values of K
i
and k
diss
shows
that oxidation also decreases the rate constant of
enzyme inhibition by factors of three to four. Thus,
the deleterious effect of elafin and trappin oxidation
on the affinity of the inhibitors for NE is caused by
both an significant increase in k
diss
and a moderate
decrease in k
ass
.
Oxidation of Met at P
1
¢ is responsible for the
decreased affinities of oxidized elafin and trappin
Elafin and the inhibitory domain of trappin each have
two methionine residues (M25 and M51 for elafin, and
M63 and M89 for trappin). M25 and M63 are the P
1
¢

residues of the inhibitors’ active centers. Mass spectro-
metry of the two proteins oxidized by N-chlorosuccini-
mide or myeloperoxidase showed that oxidation
increased the m ⁄ z by 32 Da, indicating that their two
methionine residues had been converted into methio-
nine sulfoxide (Fig. 3).
To establish which methionine residue leads to a
decrease in inhibitory activity upon oxidation, M25L
elafin and M63L trappin (two variants with a nonoxi-
dizable leucine residue at P
1
¢) were prepared. These
variants inhibited NE and porcine pancreatic elastase,
but did not react with Pr3. In addition, their affinity
for NE was lower than that observed with the wild-
type inhibitors (Table 2). Oxidation of the two vari-
ants with N-chlorosuccinimide and myeloperoxidase
increased their m ⁄ z value by 15 Da, indicating oxida-
tion of M51 and M89 of M25L elafin and M63L trap-
pin, respectively. On the other hand, oxidation of
M25L elafin and M63L trappin did not significantly
affect their K
i
for NE (Table 2). We therefore conclude
that the oxidant-promoted alteration of the K
i
of elafin
and trappin is caused by the oxidation of their P
1
¢

methionine residue.
Fig. 2. Substrate- and dilution-induced dissociation of enzyme–inhib-
itor complexes. The complexes were diluted 100-fold into a concen-
trated substrate solution ([S]
0
¼ 13.4 K
m
) and the release of
product was recorded as a function of time. (A) Neutrophil elastase
(NE)–inhibitor complexes. (B) Proteinase 3 (Pr3)–inhibitor com-
plexes. The inhibitor was N-chlorosuccinimide-oxidized trappin
(curves 1) or elafin (curves 2), and native trappin (curves 3) or elafin
(curves 4).
Fig. 3. Mass spectra of native and oxidized elafin (A) and trappin
(B). The peaks at m ⁄ z ¼ 6000.975 and 9913.063 are assigned to
the native inhibitors, whereas the peaks at m ⁄ z ¼ 6032.038 and
9948.639 are assigned to the dioxidized inhibitors.
Inhibition of elastase and proteinase 3 by elafin S. M. Nobar et al.
5886 FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS
Elastin impairs the inhibition of NE and Pr3 by
native and oxidized elafin and trappin
NE and Pr3 are both able to solubilize fibrous elastin
[19]. We used remazol-Brilliant Blue (RBB)–elastin to
investigate their elastolytic activity in the absence and
presence of native and N-chlorosuccinimide-oxidized
elafin. Preliminary experiments were designed to com-
pare the interaction of NE and Pr3 with this fibrous
substrate.
About 50% of the enzymes were immediately
adsorbed onto fibrous elastin following mixing of the

reagents and stirring. After 1 min, 70% of the enzymes
were adsorbed. Adsorption was complete after 10 min.
The affinity of elastin for NE or Pr3 was assessed by
adding a constant concentration of enzyme to increas-
ing concentrations of elastin, stirring for 10 min,
centrifugating the suspensions and measuring the
concentration of unbound enzyme using a synthetic
substrate. Both NE and Pr3 gave hyperbolic saturation
curves, as shown in Fig. 4A. Double reciprocal plots
of the data (not shown) were linear, indicating that
saturation conformed to classical reversible receptor–
ligand binding, that is R+L Ð RL (where R repre-
sents elastin and L represents NE or Pr3). The binding
curves may therefore be described by the following
equation:
[L]
bound
=[L]
total
¼ [R]
0
=ð[R]
0
þ [R]
0:5
Þð2Þ
where [R]
0
is the total concentration of elastin and
[R]

0.5
is the concentration for which 50% of enzyme
is bound. Non-linear regression analysis based on
Eqn (2) gave [R]
0.5
values of 0.77 ± 0.12 and
1.12 ± 0.25 mgÆmL
)1
for NE and Pr3, respectively.
The two enzymes therefore have similar affinities for
elastin.
To measure the elastolytic activity of enzyme ±
inhibitor mixtures, we used an elastin concentration of
3mgÆmL
)1
, which is well above the [R]
0.5
value. Under
these conditions, elastin solubilization by NE or Pr3
was linear, with time, up to an absorbance of at least
0.45 that is, up to at least 30% elastolysis (Fig. 4B).
Thus, activity measurements were very reliable. The
elastolytic activity of NE was found to be 1.9-fold
higher than that of Pr3 (Fig. 4B).
Enzyme–inhibitor mixtures were also tested in the
kinetic mode. Enzyme was added to an elastin plus
inhibitor suspension to allow it to compete between
substrate and native or oxidized inhibitors. The inhibi-
tion was assessed using either equimolecular concentra-
tions of enzyme and inhibitor, or a 10-fold molar

excess of inhibitor over enzyme. Figure 5 shows the
results of competition experiments carried out with
Table 2. Effect of M25L elafin and M63L trappin oxidation by
N-chlorosuccinimide on their affinity for neutrophil elastase (NE).
Inhibitor K
i
(M)
Elafin
Wild-type
a
0.8 ± 0.05 · 10
)10
M25L mutant 0.8 ± 0.2 · 10
)9
Oxidized M25L mutant 1.0 ± 0.1 · 10
)9
Trappin
Wild-type
a
0.3 ± 0.1 · 10
)10
M63L mutant 0.9 ± 0.2 · 10
)9
Oxidized M63L mutant 1.3 ± 0.2 · 10
)9
a
From Zani et al. [15].
Fig. 4. (A) Binding of constant concentrations of neutrophil elastase
(NE) (s) and proteinase 3 (Pr3) (h) to different concentrations of
insoluble elastin. The curves are theoretical and were generated

using Eqn (2) with [R]
0
¼ 0.77 and 1.12 mgÆmL
)1
elastin for NE and
Pr3, respectively. (B) Kinetics of solubilization of elastin by NE (D)
and Pr3 (r).
S. M. Nobar et al. Inhibition of elastase and proteinase 3 by elafin
FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS 5887
1.5 lm NE or Pr3 and 1.5 lm native or oxidized elafin.
The elastolytic activity of NE was found to be inhib-
ited much less by oxidized elafin than by the native
inhibitor (Fig. 5A), and the elastolytic activity of Pr3
was found to be almost insensitive to oxidized elafin
(Fig. 5B). Native and oxidized trappin behaved in a
similar way. With a 10-fold molar excess of inhibitor
over enzyme, we observed full inhibition of both pro-
teinases by the native inhibitors, but only % 80% inhi-
bition of NE and 50% inhibition of Pr3 by the
oxidized inhibitors.
While the above data are in overall agreement with
the results obtained using synthetic substrates, they
also indicate that elastin hinders the inhibition of
both enzymes by the native and the oxidized
inhibitors. To demonstrate this, we used Eqn (1) with
K ¼ K
i
(1 + [R]
0
⁄ [R]

0.5
) to calculate the percentage
of inhibition that would have been observed if the
system behaved like classical competitive inhibition.
Table 3 compares this theoretical inhibition with the
observed inhibition derived from the progress curves
shown, for example, in Fig. 5. It was found that (a)
the observed inhibition is lower than that with the
theoretical inhibitor, regardless of the enzyme, the
inhibitor and the state of oxidation of the latter, indi-
cating that elastin does not simply act as a competing
substrate but also hinders the inhibition process, (b)
Pr3 is much more resistant to inhibition by native ela-
fin than NE, although the two enzyme–inhibitor sys-
tems have similar kinetic constants (Table 1) and (c)
oxidized elafin and trappin are very poor inhibitors of
NE and Pr3.
Discussion
The active site of serine proteinase inhibitors is com-
posed of about eight surface-exposed amino acid resi-
dues, labeled P
5
to P
3
¢, which interact with subsites S
5
to S
3
¢ of the proteinase’s active center. S
1

–P
1
and S
1
¢–
P
1
¢ interactions play an important role in inhibitor spe-
cificity and potency [20]. In elafin ⁄ trappin, P
1
repre-
sents Ala and P
1
¢ represents Met. Oxidation of the
latter residue to methionine sulfoxide leads to a
decrease in the affinity (1 ⁄ K
i
) of the two inhibitors for
NE and Pr3. This decrease is significantly more pro-
nounced for Pr3 than for NE and is mainly the result
Fig. 5. Kinetics of solubilization of elastin by 1.5 lM neutrophil ela-
stase (NE) (A) and proteinase 3 (Pr3) (B) in the absence (D) or pres-
ence of 1.5 l
M native (h)orN-chlorosuccinimide-oxidized (s) elafin.
The order of addition of the reactants was elastin + inhibitor +
enzyme (competition experiment).
Table 3. Theoretical and observed inhibition of the elastolytic activ-
ity of neutrophil elastase (NE) and proteinase 3 (Pr3) by native
and N-chlorosuccinimide oxidized elafin and trappin. [NE] ¼ [Pr3] ¼
[elafin] ¼ [trappin] ¼ 1.5 l

M; [remazol-Brilliant Blue–elastin] ([RBB–
elastin]) ¼ 3mgÆmL
)1
. The theoretical percentage of inhibition
was calculated using Eqn (1) (competitive inhibition) with
K ¼ K
i
(1 + [R]
0
⁄ [R]
0.5
). K
i
values are from Table 1, [R]
0
is the total
concentration of elastin (3 mgÆmL
)1
) and [R]
0.5
is the elastin con-
centration at which 50% of enzyme is bound ([R]
0.5
¼ 0.77 and
1.12 mgÆmL
)1
for NE and Pr3, respectively). The observed percent-
age of inhibition is that resulting from competition experiments,
such as those shown in Fig. 5.
Enzyme Inhibitor

Percentage inhibition
Theoretical Observed
NE Elafin Native 98.4 93.0
Oxidized 87.3 25.0
Trappin Native 99.0 88.5
Oxidized 84.0 30.5
Pr3 Elafin Native 98.3 55.0
Oxidized 76.3 10.0
Trappin Native 98.0 80.0
Oxidized 70.0 19.0
Inhibition of elastase and proteinase 3 by elafin S. M. Nobar et al.
5888 FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS
of an important increase in k
diss
, the rate constant for
the dissociation of the inhibitory complexes (K
i
¼
k
diss
⁄ k
ass
). The complexes formed of native elafin or
trappin and NE or Pr3 have similar k
diss
values, which
correspond to a half-life of dissociation of 36–105 min
[15]. The oxidation of elafin and trappin down-shifts
the half-life of there complexes with NE to % 1.3–
1.8 min. On the other hand, the oxidized inhibitor–Pr3

complexes are so unstable that they relax ‘instantane-
ously’ when diluted into a substrate solution. This
means that their half-lifes are not longer than a few
seconds. The reason why oxidation renders the inhibi-
tory complexes so unstable is not clear. Methionine
sulfoxide is bulkier than methionine. Perhaps steric
hindrance prevents easy binding of the methionine
sulfoxide residue at the S
1
¢ subsite of the active centers
of NE and Pr3. The fact that the S
1
¢ subsite of Pr3 is
significantly smaller than that of NE [21] might then
explain why (a) Pr3 is more sensitive to inhibitor oxi-
dation than NE and (b) Pr3 does not react with the
Met fi Leu mutants.
Lung secretions also contain mucus proteinase inhib-
itor (SLPI), an 11.7 kDa NE inhibitor that shows
some homology with elafin [5] and whose P
1
and P
1
¢
residues are Leu and Met, respectively [22]. Oxidation
of SLPI also reduces its NE inhibitory capacity [23] as
a result of methionine sulfoxide formation [8]. Table 4
compares the kinetic properties of native and oxidized
elafin and SLPI. It can be seen that the two native
inhibitors have very close K

i
, k
ass
and k
diss
values and
that the two oxidized inhibitors also have close affinit-
ies for NE. The only difference is that the oxidation of
SLPI mainly depresses k
ass
, whereas the oxidation of
elafin mainly increases k
diss
.
Triggered neutrophils release reactive oxygen species
as well as the lysosomal enzyme, myeloperoxidase.
Therefore, the myeloperoxidase ⁄ H
2
O
2
⁄ Cl
–
system we
have used to oxidize elafin ⁄ trappin is a good model for
in vivo inhibitor oxidation in neutrophil-rich lung
inflammatory fluids. This system yields oxidized inhibi-
tors whose inhibition kinetic constants are indistin-
guishable from those observed with elafin ⁄ trappin
oxidized with N-chlorosuccinimide, the classical rea-
gent specific for surface-exposed methionine residues

[16].
Oxidation does not fully abolish the inhibitory prop-
erties of elafin and trappin. This raises the following
question: are the oxidized inhibitors still sufficiently
potent to inhibit NE and Pr3 in lung inflammation?
The in vivo potency of a proteinase inhibitor depends
upon its in vivo concentration ([I]
vivo
) and the kinetic
constants describing its inhibition of the target protein-
ase [24]. The absolute concentration of a protein in
lung secretions is difficult to measure because this pro-
tein is collected by bronchoalveolar lavage, which
dilutes it to an undefined extent. According to the rea-
soning of Ying & Simon [25], the elafin concentration
in bronchial secretions would be 1.5–4.5 lm.Ifwe
assume that an inflammatory lung secretion contains
3 lm oxidized elafin and £ 3 lm NE + Pr3 and that
there are no competing biological substrates present,
we may calculate the percentage of free enzyme using
Eqn (1) with, say, [E]
0
¼ 0.3 lm, [I]
0
¼ 3 lm and K ¼
K
i
from Table 1. This calculation shows that there is
only 0.2% free NE and 1% free Pr3 in this lung secre-
tion, indicating that, in the absence of competing sub-

strates, oxidized elafin still binds NE and Pr3 tightly.
In the lung, the situation appears to be more com-
plex: proteinases are released in a milieu that contains
both substrates and inhibitors, which may compete for
their binding. This raises the following question: are
oxidized elafin and trappin able to prevent or at least
to minimize NE- or Pr3-mediated proteolysis of insol-
uble extracellular matrix proteins, such as elastin, col-
lagen, fibronectin and laminin? We have shown that
the main effect of inhibitor oxidation is an increase in
the rate of enzyme–inhibitor complex dissociation. As
a consequence, if such a complex comes close to an
insoluble protein substrate, a fraction of enzyme may
be rapidly transferred to this substrate and proteolysis
may take place. It should be emphasized that substrate
insolubility provides high local substrate concentration
and, hence, a strong ability to dissociate an inhibitory
complex. Substrate-induced complex dissociation might
Table 4. Comparison of the effects of N-chlorosuccinimide oxidation of elafin and the mucus proteinase inhibitor (SLPI) on their interaction
with neutrophil elastase (NE).
Inhibitor K
i
(M) k
ass
(M
)1Æ
s
)1
) k
diss

(s
)1
)
Native SLPI
a
9.2 ± 2.5 · 10
)11
4.9 ± 0.5 · 10
6
4.5 ± 0.8 · 10
)4
Oxidized SLPI
a
1.1 ± 0.3 · 10
)8
2.6 ± 0.3 · 10
5
2.9 ± 0.5 · 10
)3
Native elafin
b
8.0 ± 0.5 · 10
)11
3.7 ± 0.1 · 10
6
3.2 ± 0.1 · 10
)4
Oxidized elafin
b
5.7 ± 0.6 · 10

)9
1.1 ± 0.3 · 10
)6
6.3 ± 0.6 · 10
)3
a
From Boudier & Bieth [8].
b
From Table 1.
S. M. Nobar et al. Inhibition of elastase and proteinase 3 by elafin
FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS 5889
be particularly important for the Pr3-oxidized inhibitor
complexes, whose half-life of dissociation are a few
seconds only.
We have used elastin as a substrate to verify the
above hypothesis. This insoluble polymer was able to
dissociate the native elafin–NE and elafin–Pr3 com-
plexes, despite their low K
i
and k
diss
values, confirming
the above assumption. We have used the measured
‘affinity’ of elastin for the two enzymes to calculate an
apparent K
i
, which was then used to calculate the inhi-
bition based on simple competition between substrate
and inhibitor for the binding of enzyme. This theoret-
ical inhibition was significantly higher than the experi-

mental one, again confirming the above hypothesis.
The most important differences were found for the
inhibition of Pr3 by native and oxidized elafin. The
experiments were carried out with 1.5 lm elafin, which
is within the physiological concentration range [25]. In
an equimolar mixture of enzyme and oxidized elafin,
NE and Pr3 are inhibited to the extent of 25% and
10%, respectively. This clearly shows that oxidized ela-
fin is a poor inhibitor of the elastolytic activity of these
two enzymes. Oxidized trappin is somewhat more
potent because it inhibits the two proteinases to the
extent of 30 and 19%, respectively. It may be anticipa-
ted that the oxidized inhibitors will also poorly protect
other insoluble extracellular matrix proteins from pro-
teolysis.
Inhibitor-based therapy of inflammatory lung dis-
eases has been proposed in the last decade. For
instance, aerosol-delivered a
1
-antitrypsin [26] and SLPI
[27] have been shown to augment the anti-NE capacity
of lung secretions. As elafin and trappin inhibit both
NE and Pr3, they might be potential drugs in cystic
fibrosis where enormous amounts of free NE and Pr3
are found in lung secretions [28]. However, the sensi-
tivity to biological oxidation of the wild-type inhibitors
prohibits their therapeutic use: oxidation-resistant vari-
ants must be designed. The Met ⁄ Leu variants des-
cribed here can obviously not be used because they do
not inhibit Pr3. The preparation of variants with less

bulky amino acid residues at P
1
¢ is now in progress.
Elafin is synthesized as trappin, a soluble 9.9-kDa
protein whose N-terminal cementoin domain contains
transglutaminase substrate motifs that allow it to be
covalently attached to insoluble extracellular matrix
proteins [11]. It is not unlikely that trappin forms
insoluble complexes with such proteins. Under its
insoluble form, this inhibitor might therefore be
endowed with appealing properties. First, its bioavaila-
bility might be dramatically better than that of elafin
and soluble trappin. Second, it might be more potent
than the soluble inhibitor because insolubility ‘creates’
affinity, a concept classically used in affinity chroma-
tography. Third, it might be less susceptible to oxida-
tion than the soluble molecule because insoluble
targets are more difficult to reach than soluble ones as
they do not undergo brownian motion. Hence, soluble
oxidant scavengers present in lung secretions [2] may
more efficiently protect it from oxidation. Covalently
bound trappin has not yet been identified in human
lung structures. The foregoing view is nevertheless not
pure conjecture because animal studies show that
intratracheally administered trappin (but not elafin) is
able to prevent NE-induced acute lung injury [29].
Experimental procedures
The source and active site titration of NE and Pr3 was the
same as described previously [15].
Production of recombinant M25L–elafin and

M63L–trappin
Using the elafin cDNA cloned into pGE-SKA-B ⁄ K (20 ng)
as a template [15], PCR amplification was perforrmed
according to the standard procedure of Higuchi et al. [30]
to obtain cDNAs encoding M25L–elafin and M63L–trap-
pin. For this purpose, forward primers 5¢-CGACTCGA
GAAAAGAGCGCAAGAGCCAGTCAA-3¢ and 5¢-CGAC
TCGAGAAAAGAGCTGTCACGGGAGTTCCT-3¢ were
used for amplification of the elafin and the trappin cDNA
5¢ end, respectively, and reverse primer 5¢CGAGCGGCCG
CCCCTCTCACTGGGGAAC-3¢ was used for the common
3¢ end of elafin and trappin. Oligonucleotides 5¢-GGTGCG
CCTTGTTGAATCC-3¢ (forward) and 5¢ -GGATTCAACA
AGGCGCACC-3¢ (reverse) were used to introduce the
Met ⁄ Leu substitution (Leu codon: TTG). Amplified frag-
ments were cloned into the pPIC9 vector and electroporat-
ed into P. pastoris yeast strain GS115 (his4) competent cells
(Invitrogen, Carlsbad, CA, USA).
Both recombinant inhibitors were produced and purified
by ion exchange chromatography, as described previously
for wild-type elafin and trappin [15]. Each of the molecules
migrated as a single band at 7 kDa (M25L–elafin) and
12 kDa (M63L–trappin) in a reducing SDS ⁄ PAGE gel,
indicating homogeneity of each preparation.
Oxidation of inhibitors
We used either N-chlorosuccinimide [16] or the myeloper-
oxidase ⁄ H
2
O
2

⁄ halide system [17]. In the former method,
5 lm inhibitor was reacted with 2 mm N-chlorosuccinimide
(Sigma Aldrich, Saint Quentin Fallavier, France) at pH 8.5
(200 mm Tris ⁄ HCl). After 20 min at room temperature,
0.55 vol. of the reaction medium was diluted with 0.45
vol. of 100 mm N-acetylmethionine (Bachem, Bubendorf,
Inhibition of elastase and proteinase 3 by elafin S. M. Nobar et al.
5890 FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS
Switzerland) to stop oxidation. The reaction products were
removed by gel filtration on a Sephadex G-25 column
(Pharmacia, Uppsala, Sweden), equilibrated and developed
with a 5 mm ammonium bicarbonate solution containing
3mm NaCl. The oxidized inhibitor solution was then lyo-
philized. In the latter method, 4 lm inhibitor was incubated
with 3 nm myeloperoxidase (Athens Research and Technol-
ogy, Athens, GA, USA) and 0.3 mm H
2
O
2
(VWR Interna-
tional, Fontenay Sous Bois, France) dissolved in 200 mm
sodium phosphate, 160 mm NaCl, pH 6.2. After 20 min at
room temperature, the oxidation reaction was stopped with
0.36 lm human erythrocyte catalase (Sigma, St Louis, MO,
USA). Preliminary experiments showed that the incubation
times were sufficient to obtain maximal oxidation of the
inhibitors.
Mass spectrometry
We used a Biflex MALDI-TOF spectrometer (Brucker, Wis-
sembourg, France) equipped with a reflectron and a nitro-

gen laser (k ¼ 337 nm). The samples were mixed with 1 lL
of a matrix formed of a saturated solution of a-cyano-
4-hydroxycinnamic acid in H
2
O ⁄ acetonitrile (1 : 1, v ⁄ v).
After vacuo dessication, measurements were performed in
the positive linear mode. Calibration was carried out with
insulin (m ⁄ z ¼ 5734.4) and horse heart myoglobin (m ⁄ z ¼
16952.5).
Enzymatic methods
All kinetic measurements were carried out in 50 mm Hepes,
150 mm NaCl, pH 7.4, a solution called the buffer.
The rate of solubilization of fibrous elastin was measured
using 3 mgÆmL
)1
RBB–elastin (particle size: 200–400 mesh)
(Elastin Products Co., Owensville, MO, USA) suspended in
the buffer at 37 °C. The suspension was stirred for 15 min
before the addition of enzyme, inhibitor or complex. While
stirring was continued, 500 lL samples of suspension were
withdrawn at given time-points, mixed with 500 lLof
0.75 m acetate buffer, pH 4.0, centrifuged at 10 000 g for
10 min and read at 595 nm against a blank prepared from
a reaction mixture where enzyme and inhibitor were absent.
Full solubilization of 3 mgÆmL
)1
RBB–elastin corresponds
to an absorbance at 595 nm of 1.55.
The kinetics of adsorption of NE or Pr3 to RBB–elastin
was measured by adding enzyme (final concentration

1.5 lm)to3mgÆmL
)1
substrate, withdrawing samples from
the stirred suspensions, centrifugating at 10 000 g and
adding 10 lL of supernatant to 990 lL of 0.2 mm
methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (MeOSuc-
Ala
2
-Pro-Val-pNA) or 0.29 mm methoxysuccinyl-lysyl-(2-
picolinoyl)-Ala-Pro-Val-p-nitroanilide [MeOSuc-Lys-(pico)-
Ala-Pro-Val-pNA] (Bachem) to measure the nonadsorbed
NE or Pr3, respectively. The affinity of NE or Pr3 for
RBB–elastin was measured by adding enzyme (final concen-
tration 1.5 lm) to suspensions formed of variable concen-
trations of RBB–elastin, stirring for 10 min, centrifugating
and measuring the enzymatic activities in duplicate, as des-
cribed above.
The equilibrium dissociation constant, K
i
, for the
enzyme-oxidized inhibitor complexes, was measured by
reacting increasing concentrations of oxidized inhibitors
with constant concentrations of NE (70 nm) or Pr3
(190 nm). After 20 min at 25 °C, the residual NE and Pr3
activities were measured at 410 nm and 25 °C by following
the breakdown of 0.2 mm MeOSuc-Ala
2
-Pro-Val-pNA
or 0.29 mm MeOSuc-Lys-(pico)-Ala-Pro-Val-pNA, respect-
ively. The assay times were 20–60 s. The data were fitted to

Eqn (1) [18] by nonlinear regression analysis.
The dissociation rate constant, k
diss
, of the enzyme-oxi-
dized inhibitor complexes was measured by dissociating the
complexes by both high dilution (100-fold) and high sub-
strate concentration (13.4 K
m
). A 1 lm enzyme concentra-
tion was mixed with 1 lm inhibitor in the buffer. After
30 min at 25 °C, 10 lL of this mixture was added to
990 lL of a buffered substrate solution contained in a
thermostated spectrophotometer cuvette. The substrate was
1.5 mm MeOSuc-Ala
2
-Pro-Val-pNA for the NE–inhibitor
complexes and 0.1 mm MeOSuc-Lys-(pico)-Ala-Pro-Val-
thiobenzylester [31] for the Pr3–inhibitor complexes. The
latter reaction medium also contained 3 mm 4,4¢-dithiodi-
pyridine (Sigma Aldrich), which reacts with benzylthiol to
form a complex that absorbs at 324 nm [32]. The hydro-
lysis of substrate was recorded until the absorbance varied
linearly with time, indicating that the enzyme ⁄ inhib-
itor ⁄ substrate system had reached a steady state. These
data were used to calculate the derivative curve represent-
ing the time-dependent release of free enzyme from the
inhibitory complex. The dissociation rate constant, k
diss
,
could then be calculated from this curve, as described pre-

viously [15].
Acknowledgements
We thank ‘Vaincre la mucoviscidose’, the French cystic
fibrosis foundation for financial support, Jean-Marie
Strub for mass spectrometric analysis, and Philippe
Mellet and Didier Rognan for valuable discussions.
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