Báo cáo khoa học: Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris ppt

Bieth2and Thierry Moreau1 1 INSERM U618, University Franc¸ois Rabelais, Tours, France;2Laboratory of Enzymology, INSERM U392, University Louis Pasteur, Faculty of Pharmacy, Illkirch, Fra

Kinetics of the inhibition of neutrophil proteinases by recombinant

Marie-Louise Zani1, Shila M Nobar2, Sandrine A Lacour1, Soazig Lemoine3, Christian Boudier2,

Joseph G Bieth2and Thierry Moreau1

1

INSERM U618, University Franc¸ois Rabelais, Tours, France;2Laboratory of Enzymology, INSERM U392, University Louis Pasteur, Faculty of Pharmacy, Illkirch, France;3Laboratory of Marine Biology, Universite´ Antilles-Guyane, Campus de Fouillole, Pointe a` Pitre, Guadeloupe, France

Elafin and its precursor, trappin-2 or pre-elafin, are specific

endogenous inhibitors of human neutrophil elastase and

proteinase 3 but not of cathepsin G Both inhibitors belong,

together with secretory leukocyte protease inhibitor, to the

chelonianin family of canonical protease inhibitors of serine

proteases AcDNAcoding either elafin or its precursor,

trappin-2, was fused in frame with yeast a-factor cDNAand

expressed in the Pichia pastoris yeast expression system

Full-length elafin or full-Full-length trappin-2 were secreted into the

culture medium with high yield, indicating correct processing

of the fusion proteins by the yeast KEX2 signal peptidase

Both recombinant inhibitors were purified to homogeneity

from concentrated culture medium by one-step cationic

exchange chromatography and characterized by N-terminal

amino acid sequencing, Western blot and kinetic studies

Both recombinant elafin and trappin-2 were found to be

fast-acting inhibitors of pancreatic elastase, neutrophil elastase

and proteinase 3 with kassvalues of 2–4· 106

M )1Æs)1, while dissociation rate constants kdiss were found to be in the

10)4s)1range, indicating low reversibility of the complexes The equilibrium dissociation constant Kifor the interaction

of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from kassand kdissvalues for neutrophil elastase and pro-teinase 3 Kivalues were found to be in the 10)10molar range and virtually identical for both inhibitors Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of thera-peutic potential in the treatment of various inflammatory lung diseases

Keywords: elafin; enzyme kinetics; neutrophil proteinases; Pichia pastoris; serine protease inhibitor

Inflammatory lung diseases such as chronic obstructive

pulmonary disease, emphysema, acute respiratory distress

syndrome or cystic fibrosis have been known for a long time

to be the consequence of a protease-antiprotease imbalance

The massive accumulation of stimulated

polymorpho-nuclear neutrophils (PMNs) at the site of inflammation is

accompanied by degranulation and/or lysis of these inflam-matory cells resulting in the extracellular release of a variety

of hydrolases and oxidases, as well as reactive oxygen or nitrogen species and antibacterial peptides More specifi-cally, three serine proteases including human leukocyte elastase, cathepsin G and proteinase 3, are simultaneoulsy released at high concentrations as active enzymes from azurophilic granules of activated polymorphonuclear neu-trophils where they are stored at concentrations reaching millimolar range [1,2] All three of these serine proteinases participate in the destruction of lung tissues by degrading numerous extracellular matrix proteins such as elastin, type III, IV and VI collagens, fibronectin, laminin, etc [1,3] In addition, these proteases stimulate mucous secretion by submucosal gland serous cells and goblet cells and also promote the synthesis of inflammatory cytokines, and therefore have a major role in perpetuating the inflammatory state Though other degrading proteases including metalloproteases may be released from neutrophils [e.g MMP-8 (neutrophil collagenase) and MMP-9 (92 kDa gelatinase)], it is thought that serine proteases of neutrophil origin have the greatest contribution to the protease-antiprotease imbalance observed in lung inflammation [3]

In normal lung, the proteolytic activity of extracellular neutrophil serine proteinases is efficiently regulated by at least three natural protease inhibitors present in the lung fluid, namely a-proteinase inhibitor (a-PI, also known as

Correspondence to T Moreau, INSERM U618, University Franc¸ois

Rabelais, 2bis Bd Tonnelle´, 37032 Tours Cedex, France.

Fax: + 33 247 366 046, Tel.: + 33 247 366 177,

E-mail: moreaut@univ-tours.fr

Abbreviations: a 1 -PI, a 1 -proteinase inhibitor; SLPI, secretory

leuko-cyte proteinase inhibitor; HNE, human neutrophil elastase; PR3,

human neutrophil proteinase 3; PPE, porcine pancreatic elastase;

Suc-(Ala) 3 -p-NA, succinyl-Ala-Ala-Ala-p-nitroanilide; MeO-Suc-(Ala) 2

-Pro-Val-p-NA, methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide;

MeO-Suc-Lys-(pico)-Ala-Pro-Val-TBE,

methoxysuccinyl-Lys-(2-picolinoyl)-Ala-Pro-Val-thiobenzyl ester; rec-elafin, recombinant

elafin; rec-trappin-2, recombinant trappin-2; E, enzyme;

I, inhibitor; S, substrate.

Enzymes: human neutrophil elastase (HNE; EC 3.4.21.37); human

neutrophil proteinase 3 (PR3; EC 3.4.21.76); porcine pancreatic

elastase (PPE; EC 3.4.21.36).

(Received 13 January 2004, revised 1 March 2004,

accepted 8 April 2004)

a1-antitrypsin), a member of the serpin superfamily and two

canonical, small inhibitors, secretory leukocyte proteinase

inhibitor (SLPI) and elafin In acute or chronic

inflamma-tion, the imbalance is in favor of proteases which widely

overwhelm the inhibitory capacity of lung fluid The

biological significance of this control mechanism has been

highlighted by the observation that the development of

emphysema in certain patients was related to an hereditary

deficiency of a1-PI, the major elastase inhibitor [4] In cystic

fibrosis, another inflammatory lung disease, high levels of

active neutrophil elastase, cathepsin G and proteinase 3 are

usually found in pulmonary secretions and have been

correlated with the severity of the disease [5,6] In addition

to participating to lung destruction, these proteases exhibit

various deleterious effects which contribute to maintaining

an inflammatory state and favor the persistence of microbial

infections Taken together, these observations suggest that

increasing serine protease inhibitor levels in lungs, e.g by

aerosol administration, would be beneficial to limit the

inflammation and therefore the progression of the disease

Indeed, aerosol administration of recombinant SLPI to

patients with cystic fibrosis has been shown to markedly

decrease the level of active neutrophil elastase and the

number of neutrophil at the inflammatory sites due to the

reduction of elastase-induced secretion of IL-8 [7–9] A

similar decrease in elastase levels was observed when a1-PI

was given in aerosol form to cystic fibrosis patients [10]

While development programs for recombinant SLPI have

been stalled, highly purified a1-PI produced in transgenic

animals has been obtained in huge quantities by

pharma-ceutical companies (PPL Therapeutics and Bayer), allowing

this molecule to enter clinical trials for its potential use as a

protein-based drug for cystic fibrosis As an alternative to

a1-PI, other neutrophil elastase inhibitors are currently

under development [11–13] but, like a1-PI, they target only

elastase and not the similar neutrophil proteases,

cathep-sin G or proteinase 3 We hypothesized that elafin and/or

its precursor, trappin-2 or pre-elafin, might have interesting

therapeutic potential due to their capacity to inhibit elastase

and proteinase 3 Trappin-2 is a nonglycosylated 114 amino

acid protein comprising (a) an N-terminal domain (38

residues) containing several repeated motifs with the

consensus sequence Gly-Gln-Asp-Pro-Val-Lys or

cemen-toin domain [14] that can anchor the whole molecule by

transglutaminase-catalyzed cross-links and (b) a C-terminal

four-disulphide domain (56 residues) or whey acidic

protein corresponding to elafin, that is homologous to

SLPI Elafin has been shown to be present in lung

secretions [15,16] or human epithelia [17] where it is

proteolytically released from its precursor trappin-2 by one

or several unknown protease(s) To further characterize the

maturation of elafin from trappin-2 and to compare the

antiproteolytic activity of both inhibitors, we have

expressed them in the Pichia pastoris expression system

Using a genetic construct consisting of the yeast a-factor

signal sequence, stable transformants were obtained which

secrete full-length elafin or full-length trappin-2 in the

culture media Production of elafin or trappin-2 using this

expression system allows the rapid purification of large

amounts of recombinant inhibitors which may be used for

further in vitro characterization and evaluation of their

therapeutic potential

Experimental procedures

Materials Human neutrophil elastase (HNE; EC 3.4.21.37) and human neutrophil proteinase 3 (PR3; EC 3.4.21.76) were obtained from Athens Research and Technology (Athens, USA) Porcine pancreatic elastase (PPE; EC 3.4.21.36) was purified as described previously [18] The concentrations

of active enzymes were measured according to published methods [19,20] All the enzyme or inhibitor concentrations mentioned in this article refer to active protein concen-trations Succinyl-Ala-Ala-Ala-p-nitroanilide [Suc-(Ala)3 -p-NA], methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide [MeO-Suc-(Ala)2-Pro-Val-p-NA] and methoxysuccinyl-Lys-(2-picolinoyl)-Ala-Pro-Val-thiobenzyl ester [MeO-Suc-Lys-(pico)-Ala-Pro-Val-TBE] were from Bachem

The cDNAcoding full-length trappin-2 was a kind gift of

J Schalkwijk (University of Nijmegen, the Netherlands) The pPIC9 vector was from Invitrogen (Groningen, the Netherlands) and restriction enzymes were from Life Technologies

Oligonucleotides The following primers (Genset) were used for PCR amplifications Triplets correspond to amino acids; restriction sites are underlined Primer 1; 5¢-CGA CTC GAG AAA AGA GCT GTC ACG GGA GTT CCT-3¢, restriction site XhoI This primer fuses the trappin-2 mature protein immediately downstream of the a-peptide sequence Primer 2; 5¢-CGA CTC GAG AAA AGA GCG CAAGAG CCAGTC AA-3¢, restriction site XhoI This primer fuses the elafin mature protein immediately downstream of the a-peptide sequence Primer 3; 5¢-CGAGCGGCCGCCCCTC TCACTG GGG AAC-3¢, restriction site NotI This primer corres-ponds to the common C-terminal portion of elafin and trappin-2

Primers 1 and 2 fuse the trappin-2 and elafin mature protein, respectively, immediately downstream of the a-peptide sequence and downstream of the Lys-Arg dipep-tide sequence which is removed by the yeast KEX2 protease (Pichia pastoris Expression Kit manual, Invitrogen, Groningen, the Netherlands)

Cloning of elafin and trappin-2 cDNA into pPIC9 Using the trappin-2 cDNAcloned into pGE-SKA-B/K (20 ng) as a template, PCR amplification was run for 30 cycles of 10 s at 94C, 30 s at 55 C and 45 s at 68 C with primer combination 1 & 3 or 2 & 3 All the reactions were performed using 1.5 pmol of each primer, 20 nmol of each dNTP and 1 U Taq/Pwo polymerase (Expand High Fidelity PCR system, Roche) Amplified fragments were digested with XhoI and NotI, and cloned into the pPIC9 vector The constructs containing the yeast a-peptide cDNAsequence fused to the mature elafin (pPIC9-elafin) or trappin-2 (pPIC9-trappin-2) cDNAsequence, were checked for the absence of mutations in the coding sequence by sequencing using an ABI PRISM A310 nucleotide sequencer (PE Biosystems, Courtabeuf, France)

Expression inPichia pastoris

About 10 lg of recombinant elafin or trappin-2 constructs,

previously linearized with SalI, were electroporated

(ECM399 electroporator, BTX Technologies, Hawthorne,

NY, USA) into P pastoris strain GS115 (his4) competent

cells (Invitrogen) His+ transformants were selected and

screened for elafin or trappin-2 production in small-scale

experiments For the purification of large amounts of

recombinant elafin or trappin-2, positives clones were

grown in 2 L buffered minimal glycerol-complex medium

(BMGY) at 29C for two days, harvested and suspended in

300 mL buffered minimal methanol-complex medium

(BMMY) containing 1% (v/v) methanol to induce inhibitor

production The supernatant (about 300 mL) was collected

after three (trappin-2) or seven (elafin) days of growth at

29C with constant methanol concentration (1%) and

concentrated 30-fold using a 3 kDa cutoff YM3

ultrafiltra-tion membrane (Millipore, Paris, France)

Purification of secreted elafin and trappin-2

Concentrated supernatants containing secreted elafin or

trappin-2 were dialysed over a PD10 Pharmacia column

against 25 mM sodium phosphate, pH 6.0 (equilibration

buffer) Dialysed supernatant (200 lL) was then loaded

onto a mono S column HR 5/5 (0.5 · 5 cm) equilibrated

with equilibration buffer using a Pharmacia FPLC

chro-matographic system The column was washed with 6 mL of

equilibrium buffer to eliminate unbound proteins Bound

elafin and bound trappin-2 were eluted at a flow rate of

1 mLÆmin)1 with a linear NaCl gradient of 0–0.2M in

equilibration buffer for 12 min and with a linear NaCl

gradient of 0–0.5M for 21 min, respectively Absorbance

was monitored at 220 nm The protein content of each peak

was analyzed using high resolution Tricine SDS/PAGE gels

according to Scha¨gger & von Jagow [21] After several runs

performed using the conditions described above, fractions

containing elafin or trappin-2 were pooled, concentrated by

ultrafiltration with a YM3 membrane (Millipore) and

stored at)70 C until further use The N-terminal sequence

of the purified recombinant proteins was checked using an

automated amino acid sequencer (Applied Biosystems

477A) associated with an online model 120A analyzer for

the identification of phenylthiohydantoine derivatives

Western blot analysis was performed using a goat

anti-elafin polyclonal antibody (Tebu-Bio SA, Le Perray en

Yvelines, France) according to the procedure described by

Zani et al [22]

Kinetic measurements

Stock solutions of Suc-(Ala)3-p-NAwere prepared in

N-methyl pyrrolidone Other substrates and

dithiodipyri-dine (Sigma) were prepared in dimethylformamide

Organic solvent final concentration was 1% (v/v) All

kinetic measurements were carried out at 25C in 0.05M

Hepes 0.1M NaCl, a solution referred to as the buffer

Substrate breakdown was monitored by following the

changes of absorbance at 410 or 324 nm for

para-nitroanalide or thiobenzylester derivatives, respectively

When the later substrate was used, 3 m dithiodipyridine

was present in the reaction mixtures to assess the release

of benzylthiol

Measurement of the active rec-elafin and rec-trappin-2 concentration Recombinant elafin (rec-elafin) and recom-binant trappin-2 (rec-trappin-2) preparations were active site titrated using HNE Reaction mixtures (990 lL) containing constant amounts of enzyme (0.3 lM) and increasing quantities of inhibitor were allowed to incubate for 15 min in the thermostated cell holder of a computerized Uvikon 943 spectrophotometer (Kontron Instruments, Trappes, France) before measurement of the residual enzymatic activity by addition of 10 lL of a 100 mM Suc-(Ala)3-p-NAstock solution Product release was continu-ously recorded until a constant rate of paranitroaniline production was reached (2–4 min), indicating that enzyme (E), inhibitor (I), substrate (S), and their complexes are in thermodynamic equilibrium The active concentration of both recombinant inhibitors was deduced from the volume of inhibitor necessary to totally inhibit the enzyme assuming a

1 : 1 binding stoichiometry as suggested previously [23,24] Thespecificactivityofrecombinant elafinandtrappin-2(ratio

of active inhibitor vs protein content) was found to be about 95% as inferred from active site titration experiments and determination of the protein content by the Bradford method Determination of the equilibrium dissociation constant Ki for the interaction between PPE and elafin or rec-trappin-2 Equilibrium dissociation constants governing the interaction between PPE and rec-elafin or

rec-trappin-2 were determined using titration experiments Increasing concentrations (2–25 nM) of each inhibitor were reacted in

990 lL mixtures with 10 nM elastase for 20 min, a time sufficient to ensure full enzyme–inhibitor association under the present experimental conditions as checked by prelim-inary experiments The residual enzyme activity was measured as mentioned above To check the competitive nature of the inhibition, 10 nMPPE was reacted with 10 nM rec-elafin or rec-trappin-2 in a total volume of 990 lL After

20 min, 10 lL of either 20 mMor 200 nMSuc-(Ala)3-p-NA was added to measure the residual enzyme activity Controls without inhibitor were run in parallel

Association kinetics The reactions between rec-elafin or rec-trappin-2 and PR3 or HNE were investigated using the progress curve method [25] At time zero, one volume of inhibitor + substrate solution was rapidly mixed with one volume of enzyme solution in the thermostated observation cell of a stopped flow apparatus (SFM3, Bio-Logic, Claix, France) Product formation was continuously recorded Data acquisition and analysis were performed with the BIOKINE software available from the manufacturer All experiments were done under pseudo-first order conditions, that is, with [I]0¼ 10 · [E]0

Kinetics of HNE and PR3 inhibition were studied in the presence of 1.56 mM MeO-Suc-(Ala)2-Pro-Val-p-NAand 0.15 mM MeO-Suc-Lys-(pico)-Ala-Pro-Val-TBE, respect-ively, using rec-elafin concentrations varying from 0.6 to 0.9 lM (HNE inhibition) and from 0.8 to 2.0 lM (PR3 inhibition) or rec-trappin-2 concentrations varying from 0.75 to 0.9 lM (HNE inhibition) and from 0.8 to 1.6 lM (PR3 inhibition)

Dissociation kinetics EnzymeỜinhibitor complexes were

obtained by reacting 1 lM rec-elafin or rec-trappin-2 with

the same concentration of HNE or PR3 At time 0, 10 lL

of enzymeỜinhibitor complex solution was mixed in a

spectrophotometer cuvette with 990 lL of 1.88 mM

MeO-Suc-(Ala)2-Pro-Val-p-NAor 0.20 mM

MeO-Suc-Lys-(pico)-Ala-Pro-Val-TBE in the buffer The substrate cleavage

was continuously monitored until a constant rate of

product formation was reached

Results

Expression and purification of recombinant elafin

and trappin-2

The elafin cDNAand trappin-2 cDNAwere cloned into the

yeast expression vector pPIC9, allowing the production of

both recombinant proteins in the P pastoris expression

system The cloning strategy was designed so that mature

proteins were secreted in the culture supernatant Because

both proteins have a high proportion of basic residues with

predicted pI values of 8.51 for elafin and 9.15 for trappin-2

(COMPUTE PI/MWprogram at http://www.expasy.org), no tag

was introduced for further purification of each molecule by

cation-exchange chromatography The engineered construct

contained the yeast a-peptide directly upstream the

N-terminus of either elafin or trappin-2 with a slight

modification of the linker region between the a-peptide and

the full-length protein The EAEA sequence which

corres-ponds to the yeast STE13 protease cleavage site was

removed so that the KR dipeptide was now directly

upstream of the mature elafin or trappin-2 allowing their

release by the yeast KEX2 protease Induction of protein

expression for seven days with methanol of positive yeast

clones expressing elafin resulted in a major form with a

molecular mass of 7 kDa consistent with mature elafin as

assessed by SDS/PAGE under reducing conditions and

Western blot analysis (not shown) Those conditions were

retained for large-scale production of recombinant elafin

Culture of clones expressing trappin-2 in the same

conditions followed by SDS/PAGE analysis of secreted

proteins in supernatants revealed the presence of three

pro-teins at 15, 13 and 11 kDa (Fig 1), two of which (15 kDa

and 13 kDa) were immunoreactive with antibodies

directed against elafin (not shown) N-terminal sequence

analysis indicated that full-length trappin-2

correspon-ded to the 15 kDa form whereas the 13 kDa protein

was a clipped form of trappin-2 (partial sequence:

GQDKVKAQE) resulting from a cleavage at the

K32-G33 sequence The nonimmunoreactive 11 kDa protein

was believed to be a non related yeast protein and was

not further characterized To limit the appearance of the

13 kDa clipped form of trappin-2 for the large-scale

production of trappin-2, the duration of fermentation was

reduced to three days Under these conditions, no other

proteins except the 15 kDa form corresponding to mature

trappin-2 were detected in the supernatant by SDS/PAGE

analysis (Fig 1)

Recombinant elafin and trappin-2 were purified from

yeast culture supernatants by cation-exchange

chromato-graphy as described in Experimental procedures For both

recombinant proteins, the elafin-immunoreactive material

was recovered in a single major peak (Fig 2) An aliquot from the main peak was analyzed by high-resolution Tricine SDS/PAGE which revealed a single protein of about 7 kDa and 12 kDa for elafin in reducing and nonreducing conditions, respectively, and 12 kDa (reduced) and

15 kDa (nonreduced) for trappin-2, suggesting apparent homogeneity of the purified proteins (Fig 2) N-terminal sequence analysis confirmed the identity of full-length elafin (AQEPVKGPVS) and full-length trappin-2 (AVTGVPVKGQ)

Determination of the equilibrium dissociation constant

Kifor the interaction between PPE and rec-trappin-2

or rec-elafin The equilibrium dissociation constant Kifor the interaction

of pancreatic elastase with recombinant elafin and trappin-2 was determined directly by adding substrate to an equilib-rium mixture of protease and inhibitor, and measuring spectrophotometrically the rate of release of the reaction product The concentration of both enzyme and inhibitor was low enough to obtain a concave inhibition curve [25] when incubating PPE with rec-elafin (Fig 3) The best estimates of Ki(app),the substrate-dependent Kiwas obtained

by non linear regression analysis of the data based on the following equation [25]:

a Ử1 đơE0ợ ơI0ợ Kiđappỡỡ 

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi đơE0ợ ơI0ợ K iđappỡ ỡ2 4ơE0ơI0 q

2ơE0

đ1ỡ

where a is the relative steady state rate and Ki(app)Ử

Ki(1 + [S]0/Km) The competitive nature of the inhibition was ascertained by measuring the fractional activity a for an equimolar mixture of enzyme and inhibitor using two different substrate concentrations as described in Experi-mental procedures For both inhibitors, a was found to

be substrate-dependent, indicating competitive inhibition

K was calculated from K using K Ử 1.1 mM [26]

Fig 1 Evolution of rec-trappin-2 production by Pichia pastoris as a function of the duration of fermentation Aliquots of concentrated supernatants of rec-trappin-2-secreting P pastoris cultures were ana-lyzed by high resolution SDS/PA GE and stained with Coomassie Brilliant Blue after 0, 1, 2, 3, 4, 5, 6, 7 and 10 days of fermentation (lanes d0, d1, d2, d3, d4, d5, d6, d7 and d10, respectively) Three days

of fermentation (d3) were found to be optimum for rec-trappin-2 production before unwanted proteolysis appeared, and were therefore retained for large-scale production.

Rec-trappin-2 gave a similar inhibition curve from which the Kicould be derived (not shown) The values of Kiare given in Table 1

Measurement ofkassandkdissfor the interaction

of rec-elafin or rec-trappin-2 with HNE and PR3 Linear inhibition curves were obtained when reacting increasing amounts of each inhibitor with HNE and PR3, even when using enzyme concentrations as low as 10 nM, indicating that rec-trappin-2 and rec-elafin bind both proteinases too tightly to allow the direct measurement of the equilibrium constant Ki This latter was thus calculated from the association and dissociation rate constants kass and k

Fig 2 Purification and SDS/PAGE analysis of elafin and rec-trappin-2 Aliquots (200 lL) of concentrated supernatants of rec-ela-fin- or rec-trappin-2-secreting P pastoris cultures were loaded onto a cationic exchange Mono S column After extensive washing to remove unbound proteins, bound material was eluted with a linear NaCl gradient ( -) Fractions containing purified rec-elafin (A) or purified rec-trappin-2 (B) corresponding to the major peak (shaded area) were pooled and stored at )70 C before use (C) High resolution Tricine SDS/PAGE analysis of purified elafin and purified trappin-2 under nonreducing conditions (–b) or reducing conditions (+b) Molecular masses of the protein standards are shown on the left.

Fig 3 Inhibition of the enzyme activity of pancreatic elastase by rec-elafin Constant amounts of PPE (10 n M ) were incubated for 20 min with increasing concentrations (0–2.7 · 10)8M ) of rec-elafin The residual enzymatic activity (e) was measured using Suc-(Ala) 3 -p-NA (1 m M ) as a substrate and plotted as a function of inhibitor concen-tration K i(app) was calculated by nonlinear regression analysis (Results) using these experimental points The theoretical curve (––) generated using K i(app) ¼ 1.4 n M was superimposed onto the experi-mental data Asimilar curve was obtained with rec-trappin-2.

The progress curve method was used to follow the time

course of HNE and PR3 inhibition The reagent

concen-trations were chosen to yield both easily detectable signals

and to avoid significant substrate depletion during the

acquisition time Because enzyme and inhibitor were reacted

under pseudo-first order conditions, the concentration of

product vs time is given by the following equation [25]:

½P ¼ vstþvz vs

k ð1  ektÞ ð2Þ where [P] is the product concentration at any time t, vzis the

rate of substrate breakdown at t¼ 0 and vs the steady state

rate The best estimates of k, the apparent pseudo-first order

rate constant for the approach to the steady state, vzand vs

were obtained by non linear regression analysis of the

progress curves based on Eqn (2) HNE and PR3 inhibition

was analysed by assuming that E and I react according to

a bimolecular and reversible mechanism as described in

Scheme I

Hence, kass, kdiss and Ki may be deduced from the

following relationships [25]:

k¼ kass½I0

1þ ½S0=Km

Ki¼ kdiss=kass ð5Þ Kinetics for the association of HNE and Pr3 with rec-elafin

or rec-trappin-2 were studied as described in Experimental

procedures We observed good fits of the experimental data

to the theoretical curves generated using the best estimates of

k, indicating that enzyme inhibition was satisfactorily described by Eqn (2) (not shown) Also, k was proportional

to [I]0 Typical values of k were 0.33 ± 0.02 s)1 and 0.19 ± 0.02 s)1for the association of HNE with 0.9 lM rec-elafin and rec-trappin-2, respectively, 0.16 ± 0.01 s)1 for the reaction of PR3 with 0.8 lM rec-elafin and 0.19 ± 0.02 s)1for the inhibition of PR3 by 1.6 lM rec-trappin-2

Accurate values of kdiss could not be calculated using Eqn (4) because of the almost complete inhibition of HNE and PR3 once the steady state was reached For this reason, the dissociation rate constant was independ-ently obtained from further experiments Figure 4Ashows the kinetics of product accumulation following the dilution of an aliquot of preformed HNE–rec-elafin complex into substrate solution Complex dissociation was triggered by both high dilution (100-fold) and high substrate concentration ([S]0¼ 13.4 Km) The concentra-tion of the latter was appropriate to ensure both sufficient dissociation (Scheme I) and continuous enzyme detection without significant decrease of its concentration during the experiment The experimental data were used to calculate the derivative curve (Fig 4B) representing the concentra-tion of free enzyme vs time Free enzyme was almost absent at t¼ 0 and its concentration increased up to a steady state level corresponding to 17% of the total enzyme present in the reaction mixture (1.7 nM), indica-ting that E, I and S were in thermodynamic equilibrium with their complexes Asimilar procedure was used to study the dissociation kinetics of 1 lMHNE–rec-trappin-2, PR3–rec-elafin and PR3–rec-trappin-2 complexes Their 100-fold dilution into the appropriate substrate solutions yielded 12%, 43% and 46% of total enzyme release, respectively

As neither free enzyme nor free inhibitor were present to a significant extent at t¼ 0, the rate of complex dissociation, that is, the rate of enzyme release, is given by:

d½EI

dt ¼d½E

dt ¼ kass½EI  kdiss½E½I ð6Þ which integrates into Eqn (7) [27]:

½E ¼ ½Ee ðefk diss tð2½EI0½EeÞ=½Eeg 1Þ

efk diss tð2½EI0½EeÞ=½Eeg ½Ee=½EI þ 1 ð7Þ where [E]eand [E] are the concentrations of free HNE or PR3 at equilibrium and at any time t, respectively, and [EI]0 and [EI] are the initial concentration of complex and its

Scheme 1.

Table 1 Equilibrium and rate constants for the inhibition of neutrophil elastase, proteinase 3 and pancreatic elastase by recombinant elafin and recombinant trappin-2 Methods and experimental conditions are described in Experimental procedures Values are given as means ± SEM ND, not determined.

Enzyme

k ass ( M )1 Æs)1) k diss (s)1) K i ( M ) k ass ( M )1 Æs)1) k diss (s)1) K i ( M ) Neutrophil elastase (3.7 ± 0.1) 10 6 (3.2 ± 0.1) 10)4 (0.8 ± 0.05) 10)10a (3.6 ± 0.5) 10 6 (1.1 ± 0.2) 10)4 (0.3 ± 0.1) 10)10a Proteinase 3 (3.3 ± 0.03) 106 (4 ± 0.3) 10)4 (1.2 ± 0.1) 10)10a (2 ± 0.1) 106 (3.7 ± 1.1) 10)4 (1.8 ± 0.6) 10)10a Pancreatic elastase ND ND (7.5 ± 1.5) 10)10 ND ND (3.2 ± 0.8) 10)10

a Calculated as the k diss /k ass ratio.

concentration at any time t, respectively Figure 4B shows

the theoretical curve calculated by non linear regression

analysis of the data using Eqn (7) and using the best

estimate of kdissgoverning the dissociation of the

HNE–rec-elafin complex Good fits were also obtained for the three

other enzyme–inhibitor pairs indicating that the kinetics of

enzyme release satisfactorily agrees with Eqn (7)

Compar-ison of the kdissvalues found using this procedure (Table 1)

with values of k reported above show that kdissis

400–1700-fold lower than k and may therefore be neglected in Eqn (3)

The average association rate constant kassfor each

enzyme-inhibitor pair were therefore calculated from kass¼

k (1 + [S]0/Km)/[I]0 and [S]0¼ 1.56 mM, Km¼ 0.14 mM

for the HNE-MeO-Suc-(Ala)2-Pro-Val-p-NAsystem [28]

and [S]0¼ 0.15 mM, Km¼ 0.01 mM for the

PR3-MeO-Suc-Lys-(pico)-Ala-Pro-Val-TBE pair [29] These kassvalues

are reported in Table 1

Discussion

The control of the excessive proteolytic activity of HNE has long been recognized to be crucial to avoid degradation of the lung parenchyma in many inflammatory lung diseases

As a consequence, lung therapies based on the inhibition of HNE have lead to intensive research on the development of HNE inhibitors, either as recombinant proteins or synthetic small-molecule inhibitors [13] However, there is concern now that other neutrophil-derived proteases, namely cath-epsin G and proteinase 3, might have similar deleterious effects as HNE, hence the necessity to design inhibitors able

to target all three neutrophil-derived serine proteases In our efforts to evaluate the therapeutic potential of recombinant genetically modified protease inhibitors derived from nat-ural inhibitors, we report here on the biosynthetic produc-tion of elafin and its precursor, trappin-2 The cDNA coding either elafin or trappin-2 was cloned into the yeast expression vector pPIC9, allowing the production of both inhibitors in the P pastoris system The cloning strategy was designed so that mature elafin or mature trappin-2 were secreted in the culture supernatant No tag to facilitate the purification of the expressed proteins was introduced because both proteins were predicted to be mainly basic, allowing further purification with cation-exhange chroma-tographic procedures While the level of elafin production increased up to seven days of fermentation with no apparent modifications of the protein, the expression of trappin-2 was found to become sensitive to unwanted proteolysis as fermentation duration increased Aclipped form of trappin-2 resulting from a cleavage C-terminal to Lys32 appeared together with the full-length trappin-2 after three days of fermentation Such a proteolytic susceptibility after lysyl residues was observed by Bourbonnais et al [30] who expressed trappin-2 in Saccharomyces cerevisae Clea-vage after Lys14 and Lys36 in the so-called cementoin domain of trappin-2 was attributed unambiguously to yapsin-1, an aspartic plasma membrane protease active within the periplasmic space Though the cleavage sites in trappin-2 were different in the two yeast expression systems,

we can hypothesize that yapsin-like enzyme(s) are also involved in the non specific degradation of heterologous proteins expressed in P pastoris Reducing the fermentation

to a maximum of three days for yeast clones expressing trappin-2 was found to suppress the apparition of the

13 kDa clipped form of trappin-2 at the cost of a somewhat lower protein concentration Using the culture conditions described above, we purified about 15 mgÆL)1 of each recombinant inhibitor from the yeast culture media Using shake-flask culture conditions which give expression levels typically low relative to what is obtainable in fermenter cultures [31], we found that the amount of elafin and trappin-2 produced in our system was higher than that reported for trappin-2 expressed in similar conditions in the yeast S cerevisiae system (2–3 mgÆL)1) [30] Though the range of expression yields is variable from one protein to another, our study confirms that the P pastoris system allows the production of heterologous proteins at a high concentration level In addition, considering the ease by which the protein production can be scaled up from shake-flask to fermentation conditions [31], P pastoris is a system

of choice to produce large amounts of therapeutic proteins

Fig 4 Dissociation kinetics of HNE–rec-elafin complexes (A) Time

course of p-nitroaniline release resulting from the hydrolysis of

MeO-Suc-(Ala) 2 -Pro-Val-p-NAby HNE released from its complex with

rec-elafin Complexes were first formed by incubating equimolar

concentrations (10)6M ) of enzyme and inhibitor Dissociation of the

complexes was induced by dilution in a concentrated substrate

(1.88 m M ) solution (B) Kinetics of elastase release calculated from

(A) as described in Results The theoretical curve (––) superimposed

onto the experimental data was calculated using Eqn (7) and k diss ¼

3.2 10)4s)1.

N-terminal sequencing and Western blot analysis showed

that recombinant elafin and trappin-2 are identical to the

natural proteins In addition, enzyme kinetics showed that

the Ki of PPE–rec-elafin complex is very close to that

reported for natural elafin [32] Also, the kinetic constants

kass, kdissand Kifor the interaction of rec-elafin with HNE

and PR3 are of the same order of magnitude as those

reported for chemically synthesized elafin by Ying & Simon

[23,24] The P pastoris expression system described here

therefore yields a protein structurally and functionally

identical to natural elafin

Litterature lacks information on the kinetic parameters

describing the interaction of trappin-2 with HNE and

PR3 Based on the kinetic parameters determined here,

the most important result of our investigation is that

elafin and trappin-2 have very close inhibitory capacities

This means that the N-terminal cementoin domain of

trappin-2 has little or no influence on the reactive

inhibitory site of elafin However, it is noteworthy that

trappin-2, but not elafin, has been shown to significantly

reduce a HNE-induced experimental lung hemorrhage in

hamsters [33] or a lipolysaccharide-induced acute lung

inflammation in mice [34] This has been attributed to the

unique capacity of the cementoin domain to be

cross-linked to extracellular matrix proteins through the

cata-lytic action of tissue transglutaminase(s) [33,34] In this

context, it will be especially interesting to evaluate the

inhibitory properties of bound trappin-2, as this covalent

linking may increase significantly the bioavailability of

such an inhibitor at the site of inflammation, e.g in the

case of therapeutic administration, as well as providing a

source of inhibitory elafin

Knowledge of the kinetic parameters characterizing a

protease–inhibitor interaction and of the in vivo

concen-tration of an inhibitor is necessary to evaluate whether

such an inhibitor may control the activity of its target

enzyme(s) [25,35] From the kinetic constants determined

here and from the in vivo concentration of elafin estimated

to be in the range 1.5–4.5 lM in bronchial secretions of

normal patients [16,24], we can conclude that both elafin

and its precursor are fast-acting inhibitors of HNE and

PR3 with a delay time for total inhibition of a few

milliseconds (d(t)¼ 5/kassÆ[I]0[35]) The second conclusion

is that both inhibitors will exhibit a pseudo-irreversible

behaviour because the [I]0/Kiratio of about 15–45· 103is

greater than 103 [25]

Altogether, our results clearly demonstrate for the first

time that, in vitro, trappin-2 and elafin exhibit a similar and

potent inhibitory capacity towards HNE and PR3, strongly

suggesting that boosting elafin or trappin-2 level by an

aerosol administration would be beneficial in the treatment

of inflammatory lung diseases

Acknowledgements

This study was supported by the French cystic fibrosis association

Vaincre la Mucoviscidose We thank Dr Antoine Touze´ for nucleotide

sequencing, Miche`le Brillard-Bourdet for N-terminal protein

sequen-cing and Prof Joost Schalkwijk and Dr Patrick Zeeuwen for their kind

gift of trappin-2 cDNA We also thank Dr Fre´de´ric Delamotte and

Prof Francis Gauthier for valuable discussions Shila M Nobar holds

a fellowship from Vaincre la Mucoviscidose.

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