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.. To express the oxidation effe
Trang 1activity of neutrophil elastase and proteinase 3
Shila M Nobar1, Marie-Louise Zani2, Christian Boudier1, Thierry Moreau2and Joseph G Bieth1
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
H2O2, 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: jgbieth@pharma.u-strasbg.fr
(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⁄ H2O2 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 (Ki¼ 4.0–9.2 · 10)9m) and for Pr3 (Ki¼ 2.5– 5.0· 10)8m) 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.
Trang 2cathepsin 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 a1
-protein-ase inhibitor (also called a1-antitrypsin; a 53-kDa
pro-tein that inhibits the above three enzymes) [3];
a1-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 (Eþ I !kass
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 Eþ I Ðkass
k diss
EI an equilibrium dissociation constant
Ki¼ kdiss⁄ kass[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 kass¼ 2–4 · 106m)1Æs)1 and
formed very stable inhibitory complexes with kdiss and
Kiin the 10)4Æs)1and 10)10mrange, 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 myeloperoxidase⁄
H2O2⁄ 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 + NE or Pr3; (s), (D), oxidized inhibitors + NE or Pr3, respectively.
Trang 3Pr3 Straight inhibition curves were obtained with the
native inhibitors, in agreement with the low values of
Ki [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, Ki, for the complexes formed of oxidized elafin
or trappin and NE or Pr3 Oxidation was carried out
with either N-chlorosuccinimide or myeloperoxidase
The Ki values were determined from inhibition curves,
such as those shown in Fig 1 These curves were
ana-lyzed using Eqn (1):
aỬ 1 đơE0ợ ơI0ợ Kỡ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi đơE0ợ ơI0ợ Kỡ2 4ơE0ơI0 q
2ơE0
đ1ỡ 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 KỬ Ki if the substrate (S) does not
dissociate EI during the 20Ờ60 s assay of enzymatic
activity or KỬ Ki(1+[S]0⁄ Km) 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 inhibitorỜNE complex was slow enough to
be insignificant during the 20Ờ60 s time period used to
measure the activities of the inhibitory mixtures
Therefore, the K of Eqn (1) is substrate-independent and equals Ki In contrast, dissociation of the oxidized inhibitorỜPr3 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 Ki(1 + [S]0⁄ Km) As shown in Table 1, oxidation of elafin and trappin sig-nificantly increases the Ki (decreases the affinity) for its complexes with NE and Pr3 Oxidation by N-chloro-succinimide or myeloperoxidase yields inhibitors whose Ki 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 Ki caused by an increase in the dissociation rate constant, kdiss, a decrease in the association rate constant, kass, or an effect on both parameters (KiỬ kdiss⁄ kass)? To answer this question, we measured kdissby extensively diluting equimolar enzymeỜinhibitor 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 enzymeỜ inhibitorỜsubstrate 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 kdissand Kivalues are experimental, whereas the kassvalues are calculated MPO, myeloperoxidase ⁄ H 2 O 2 ⁄ Cl Ờ
; NCS, N-chlorosuccinimide; ND, not determined.
diss (s)1)
a Calculated assuming that dissociation is terminated in 30 s or less, which corresponds to a t ơ ặ 6 s.
Trang 4Quantitative calculation of kdiss 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, kdiss 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
kdissof 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 kass for the NE-oxidized elafin and trappin
com-plexes using the measured values of Kiand kdiss 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 kdiss and a moderate decrease in kass
Oxidation of Met at P1¢ 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 P1¢ 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 P1¢) 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 Kifor NE (Table 2) We therefore conclude that the oxidant-promoted alteration of the Kiof elafin and trappin is caused by the oxidation of their P1¢ 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]0¼ 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.
Trang 5Elastin 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, stirrincreas-ing 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+LÐ RL (where R
repre-sents elastin and L reprerepre-sents NE or Pr3) The binding
curves may therefore be described by the following
equation:
[L]bound=[L]total¼ [R]0=ð[R]0þ [R]0:5Þ ð2Þ
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]0.5values 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
3 mgÆmL)1, which is well above the [R]0.5value 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).
Elafin
Trappin
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]0¼ 0.77 and 1.12 mgÆmL)1elastin for NE and Pr3, respectively (B) Kinetics of solubilization of elastin by NE (D) and Pr3 (r).
Trang 61.5 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% inhiinhi-bition 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
K¼ Ki(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-excom-posed amino acid resi-dues, labeled P5 to P3¢, which interact with subsites S5
to S3¢ of the proteinase’s active center S1–P1 and S1¢–
P1¢ interactions play an important role in inhibitor spe-cificity and potency [20] In elafin⁄ trappin, P1 repre-sents Ala and P1¢ represents Met Oxidation of the latter residue to methionine sulfoxide leads to a decrease in the affinity (1⁄ Ki) 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 l M neutrophil
ela-stase (NE) (A) and proteinase 3 (Pr3) (B) in the absence (D) or
pres-ence of 1.5 l M native (h) or N-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 3 mgÆmL)1 The theoretical percentage of inhibition was calculated using Eqn (1) (competitive inhibition) with
K ¼ K i (1 + [R]0⁄ [R] 0.5 ) Kivalues are from Table 1, [R]0is the total concentration of elastin (3 mgÆmL)1) and [R]0.5is the elastin con-centration at which 50% of enzyme is bound ([R] 0.5 ¼ 0.77 and 1.12 mgÆmL)1for 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
Trang 7of an important increase in kdiss, the rate constant for
the dissociation of the inhibitory complexes (Ki¼
kdiss⁄ kass) The complexes formed of native elafin or
trappin and NE or Pr3 have similar kdissvalues, 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 S1¢ subsite of the active centers
of NE and Pr3 The fact that the S1¢ 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
Metfi Leu mutants
Lung secretions also contain mucus proteinase
inhib-itor (SLPI), an 11.7 kDa NE inhibinhib-itor that shows
some homology with elafin [5] and whose P1 and P1¢
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 Ki, kassand kdiss 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 kass, whereas the oxidation of
elafin mainly increases kdiss
Triggered neutrophils release reactive oxygen species
as well as the lysosomal enzyme, myeloperoxidase
Therefore, the myeloperoxidase⁄ H2O2⁄ 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 If we 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]0¼ 0.3 lm, [I]0¼ 3 lm and K ¼
Ki 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).
diss (s)1)
4.5 ± 0.8 · 10)4
a From Boudier & Bieth [8] b From Table 1.
Trang 8be 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 Kiand kdissvalues, confirming
the above assumption We have used the measured
‘affinity’ of elastin for the two enzymes to calculate an
apparent Ki, 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 a1-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 P1¢ 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
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⁄ H2O2⁄ 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,
Trang 9Switzerland) 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
3 mm 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 H2O2(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 (k¼ 337 nm) The samples were mixed with 1 lL
of a matrix formed of a saturated solution of
a-cyano-4-hydroxycinnamic acid in H2O⁄ 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)1RBB–elastin (particle size: 200–400 mesh)
(Elastin Products Co., Owensville, MO, USA) suspended in
the buffer at 37C 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 lL of
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) to 3 mgÆmL)1substrate, 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-Ala2-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
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, Ki, 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 25C, the residual NE and Pr3 activities were measured at 410 nm and 25C by following the breakdown of 0.2 mm MeOSuc-Ala2-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, kdiss, 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 Km) A 1 lm enzyme concentra-tion was mixed with 1 lm inhibitor in the buffer After
30 min at 25C, 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-Ala2-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, kdiss, 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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