Báo cáo khoa học: Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L docx

Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L Claire Desmazes1, Laurent Galineau1, Francis Gauthier1, Dieter Bro¨mme2and Gil

Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L

Claire Desmazes1, Laurent Galineau1, Francis Gauthier1, Dieter Bro¨mme2and Gilles Lalmanach1

1

Laboratoire d’Enzymologie et Chimie des Prote´ines, Equipe Prote´ases et Vectorisation, INSERM EMI-U 00 10,

Universite´ Franc¸ois Rabelais, Faculte´ de Me´decine, Tours, France; 2Department of Human Genetics,

Mount Sinai School of Medicine, New York, USA

Macrophages at an inflammatory site release massive

amounts of proteolytic enzymes, including lysosomal

cys-teine proteases, which colocalize with their circulating,

tight-binding inhibitors (cystatins, kininogens), so modifying the

protease/antiprotease equilibrium in favor of enhanced

proteolysis We have explored the ability of human

cath-epsins B, K and L to participate in the production of kinins,

using kininogens and synthetic peptides that mimic the

insertion sites of bradykinin on human kininogens

Although both cathepsins processed high-molecular weight

kininogen under stoichiometric conditions, only

cathep-sin L generated significant amounts of immunoreactive

kinins Cathepsin L exhibited higher specificity constants

(kcat/Km) than tissue kallikrein (hK1), and similar Michaelis

constants towards kininogen-derived synthetic substrates A

20-mer peptide, whose sequence encompassed kininogen residues Ile376 to Ile393, released bradykinin (BK; 80%) and Lys-bradykinin (20%) when incubated with cathep-sin L By contrast, cathepcathep-sin K did not release any kinin, but a truncated kinin metabolite BK(5–9) [FSPFR(385– 389)] Accordingly cathepsin K rapidly produced BK(5–9) from bradykinin and Lys-bradykinin, and BK(5–8) from des-Arg9-bradykinin, by cleaving the Gly384-Phe385 bond Data suggest that extracellular cysteine proteases may par-ticipate in the regulation of kinin levels at inflammatory sites, and clearly support that cathepsin K may act as a potent kininase

Keywords: cathepsin; cysteine protease; inflammation; kinin; kininogen

Kinins, whose archetype is bradykinin (BK), are generated physiologically from kininogens by tissue and plasma kallikreins [1,2], and their pharmacological effects mediated either by inducible (B1-type) or constitutive (B2-type) kinin receptors [3] In addition to their physiological role, kinins are implicated in inflammatory disorders, causing vasodil-atation and contraction of smooth muscles; they also stimulate the release of nitric oxide, increase microvascular permeability and modulate the release of histamine, prostaglandine E2, superoxide radicals and pro-inflamma-tory cytokines, IL-1 and TNF-a [4–7] Since the character-ization of BK [8], other kinins have been identified, including kallidin (Lys-BK), and des-Arg9-BK Plasma kallikrein forms bradykinin from high-molecular weight kininogen, whereas tissue (glandular) kallikrein forms kallidin from low- and high-molecular weight kininogens (LMWK/HMWK) Their amount is regulated by kininases, which rapidly breakdown kinins to give peptidyl fragments, some of which remain pharmacologically active [2] Mast cells, neutrophils, and macrophages all migrate to the site of injury during chronic or acute inflammation Macrophages secrete cytokines, oxygen radicals and pro-teolytic enzymes in addition to killing cells and carrying out phagocytosis [9] This is especially true for inflammatory lung diseases (asthma, COPD and emphysema) where the protease/antiprotease balance appears to be tipped in favour of enhanced proteolysis, due to an increase in proteases (neutrophil elastase, cathepsins, matrix metallo-proteinases), or the partial inactivation and/or lack of antiproteases (such as a1-proteinase inhibitor, elafin, secre-tory leukocyte protease inhibitor), favoring the destruction

Correspondence to G Lalmanach, Laboratoire d’Enzymologie et

Chimie des Prote´ines, INSERM EMI-U 00-10, Universite´ Franc¸ois

Rabelais, Faculte´ de Me´decine, 2 bis, Boulevard Tonnelle´,

37032 Tours cedex, France.

Fax: +33 2 47 36 60 46, Tel.: +33 2 47 36 61 51,

E-mail: lalmanach@univ-tours.fr

Abbreviations: Abz, ortho-aminobenzoic acid; ACE,

angiotensin-converting enzyme; AMC, 7-amino-4-methyl-coumarin

hydrochlo-ride; BAL, bronchoalveolar lavage; BK, bradykinin; C-BK,

C-terminal bradykinin-derived substrate; CP, cysteine protease(s);

COPD, chronic obstructive pulmonary disease; DTT, DL

-dithiothrei-tol; E-64, L

-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanido)butane; hK1, human tissue kallikrein; HMWK,

high-molecular weight kininogen; IL-1, interleukin-1; L -BAPA,

Na-benzoyl- L -arginine-4-nitroanilide; LMWK, low-molecular weight

kininogen; Lys-BK, kallidin; N-BK, N-terminal bradykinin-derived

substrate; 3-NO 2 -Tyr, 3-nitro-tyrosine; PCMPSA,

p-chloromercuri-phenylsulfonic acid; Rink amide MBHA resin,

(4-(2¢,4¢-dimethoxy-phenyl-Fmoc-aminomethyl-phenoxyacetamido-norleucyl)-4

methylbenzhydrylamine) resin; TNF-a, tumor necrosis factor-a;

Z, benzyloxycarbonyl.

Enzymes: Human cathepsin K (EC 3.4.22.38); human cathepsin B

(EC 3.4.22.1); human cathepsin L (EC 3.4.22.15); papain

(EC 3.4.22.2); human tissue kallikrein (EC 3.4.21.35); bovine

pancreatic trypsin (EC 3.4.21.4).

(Received 8 August 2002, revised 24 October 2002,

accepted 20 November 2002)

of connective tissue and the spread and severity of

inflammation [10–12] Although Travis et al have suggested

that kallikreins may loose their ability to operate in vivo at

inflammatory sites [13], local kinin production seems to be

undisturbed Kinins may be produced by other pathways

involving the concerted action of two trypsin-like serine

proteases, as already shown for the kallikrein–human

neutrophil elastase couple [14], or the tryptase–human

neutrophil elastase couple [13] Alternatively, there is

growing evidence that lysosomal cysteine proteases (CP)

are released from macrophages during lung inflammation

and colocalized with their natural inhibitors, cystatins and

kininogens [15–18] We suggested recently that human

cathepsin L may generate kinins from LMWK and

HMWK [19] The activity and stability of extracellular CP

may be favored by the increased expression of vacuolar-type

H+-ATPase components, which reduces the pH of the

pericellular environment of macrophages [20] This raises

the question of the dual behaviour of kininogens complexed

with CP While concentrations of cystatins C, S, SA and SN

are decreased [21] and cystatin C is inactivated by

neutro-phil elastase [22], recent pharmacokinetic studies have

demonstrated that the distribution of HMWK throughout

the body, that is concentrated mostly in lung, is correlated

with BK metabolism and activity [23] Taken together, these

data point to the disruption of the cathepsin/cystatin

balance, during lung inflammation

In our efforts to characterize the proteolytic activity of

inflammatory bronchoalveolar lavage fluids, we found that

massive amounts of active lysosomal CP were released from

macrophages, leading to a des-equilibrium of the cystatin/

CP balance in favor of enzymes, while kininogens

were highly degraded (C Serveau, M Ferrer-Di Martino,

and G Lalmanach, unpublished observations) Based on

these observations, the aim of the present report was to

explore the ability of cathepsins B, L and K to process

kininogens and participate to the kinin metabolism In vitro

kinetic studies were performed using native kininogens and

fluorogenic kininogen-derived peptides as models, in order

to identify and quantify the peptides released (kinins or their

kinin-like moities)

Experimental procedures

Materials

Z-Phe-Arg-AMC and dithiothreitol was purchased from

Bachem Biochimie (Voisins-le-Bretonneux, France)

Na-benzoyl-L-arginine-4-nitroanilide (L-BAPA) was from

Merck KgaA (Darmstadt, Germany) E-64 and

phenyl-methanesulfonylfluoride were from Sigma-Aldrich (St

Quentin le Fallavier, France) Molecular mass calibration

kits were from Bio-Rad (Ivry-sur-Seine, France) All other

reagents were of analytical grade

Enzymes

Human cathepsin K (EC 3.4.22.38) was prepared as

repor-ted previously [24] Human cathepsin B (EC 3.4.22.1) and

human cathepsin L (EC 3.4.22.15) were supplied by

Cal-biochem (France Biochem, Meudon, France) Papain

(EC 3.4.22.2) was obtained from Boehringer (Roche

Molecular Biochemicals, Mannheim, Germany) The activa-tion buffer for cathepsins and papain was 0.1Mphosphate buffer, pH 6.0 (containing 2 mM dithiothreitol and 1 mM EDTA) Enzymes were activated in their assay buffer for

5 min at 37C prior to the making of kinetic measurements (spectrofluorimeter Kontron SFM 25) Their active sites were titrated with E-64 [25], using Z-Phe-Arg-AMC as the substrate (excitation wavelength: 350 nm; emission wave-length: 460 nm) Human tissue kallikrein (EC 3.4.21.35) was obtained from Sigma-Aldrich, while bovine pancreatic trypsin (EC 3.4.21.4) was purchased from Roche Molecular Biochemicals The buffer for trypsin was 0.1M Tris/HCl,

pH 8.5, containing 0.15M NaCl, and that for hK1 was

50 mMTris/HCl, pH 8.3, containing 1 mMEDTA Trypsin and hK1 were titrated as reported elsewhere [26]

Inhibitors Human low- and high-molecular weight kininogens were purchased from Calbiochem Rat T-kininogen (also called thiostatin) was prepared as reported previously [27] Both kininogens were titrated with E-64-titrated commercial papain [25]

Peptides Unless otherwise stated, all Fmoc-protected amino acids were of the L-configuration, and were purchased from Neosystem (Strasbourg, France) or Advanced Chemtech (Cambridge, UK) N-BK peptide, C-BK peptide and BK-peptide were prepared by Fmoc chemistry on an automated solid phase peptide synthesizer (ABI model 431 A, Applied Biosystems, Roissy, France), using a Rink Amide MBHA resin (Novabiochem) After removal of the side chain protecting groups and cleavage from the resin, peptidyl amides were purified by semipreparative reverse phase chromatography (Vydac C18218TPS1 column), using a 35-min linear (0–60%) gradient of acetonitrile in 0.1% trifluoroacetic acid Finally, the peptides were checked for homogeneity by analytical RP-HPLC (Brownlee C18

OD 300 column), using the elution conditions indicated above, and their molecular weights checked by MALDI-TOF MS (Bru¨ker) An aliquot of the N-BK peptide (0.1 mM) was incubated with aqueous N-chlorosuccinimide (ICN Pharmaceuticals, Orsay, France) (5 mM) in 0.1MTris/HCl buffer, pH 8.5, for 1 h at room temperature to oxidize the methionyl group (Met379) The oxidized peptide was purified

by RP-HPLC (Vydac C18218TPS1 column), using a 35-min linear (0–60%) gradient of acetonitrile in 0.1% trifluoroacetic acid Presence of methionine sulfoxide at position 379 was controlled by mass spectroscopy The bradykinin-derived pentapeptidylamide BK(5–9) (FSPFR) was prepared by solid phase synthesis as described above, while bradykinin (BK) and des-Arg9-BK were obtained from Sigma-Aldrich, and Lys-BK was from Advanced Chemtech

Proteolysis of HMWK by cathepsins HMWK (0.55 lM) was incubated with different concentra-tions of cathepsins B, L and K at kininogen/enzyme ratios

of 4 : 1, 2 : 1 and 1 : 1 (two cystatin-like inhibitory sites per kininogen) in 0.1MNaCl/P, pH 6.0, 1 mMEDTA, 2 mM

dithiothreitol for 60 min at 37C The reaction was stopped

by adding SDS/PAGE sample buffer Samples were boiled

for 3 min and subjected to SDS/PAGE 10% under reducing

conditions [28] A control experiment was performed using

the same procedure, except that the HMWK was incubated

with hK1 at enzyme/kininogen ratios of 1 : 10 and 1 : 100 in

50 mMTris/HCl buffer, pH 8.3, EDTA 1 mMfor 60 min at

37C

Kinetics measurement

Determination of kcat/Km The second-order rate

con-stants for the hydrolysis of fluorogenic substrates by

cathepsins B, L and K, and of hK1 were determined under

pseudo-first order conditions (Hitachi F-2000

spectro-fluorimeter; excitation wavelength: 320 nm; emission

wave-length: 420 nm), and calibration was performed as

described elsewhere [29] Assays (in triplicate) were carried

out by adding cathepsins K (4 nM),B (4 nM), or L (2 nM) or

hK1 (4 nM) to N-BK peptide (final concentration: 0.5 lM)

Kinetic data were determined using theENZFITTERsoftware

(Biosoft, Cambridge, UK) and are reported as

means ± SD [30] The second-order rate constants for

hydrolysis of C-BK peptide were determined under the

same experimental conditions, except for cathepsins K

(6.7 nM) and L (6 nM)

Determination of the Michaelis constant (Km) Kmvalues

were determined from Hanes linear plots, with various

concentrations of C-BK peptide (1–10 lM) plus

cathep-sins L (6 nM), K (4 nM) and B (3.7 nM), and hK1 (4 nM)

The Michaelis constants for hydrolysis of N-BK peptide by

hK1 and cathepsin B were determined similarly, while the

Kmfor cathepsins L and K were determined under mixed

alternative substrate conditions, according to Segel [31],

using L-BAPA as chromogenic substrate Under these

conditions, each substrate acted as a competitive inhibitor

of the other (Eqn 1), and the Kmvalues for the fluorogenic

substrate were obtained by measuring the dissociation

constant (Ki) towards the chromogenic substrate Assays

were carried out by adding cathepsin L (60 nM) or

cathepsin K (60 nM) to a mixture ofL-BAPA (50–750 lM)

(whose Kmare 86 lMand 66 lM, respectively) and N-BK

peptide (1–10 lM) [32] The hydrolysis of L-BAPA was

monitored at 410 nm (Hitachi U-2001 spectrophotometer),

with less than 5% ofL-BAPA hydrolyzed The velocity of

the reaction is described by:

vi=vo ¼ fðKmþ SÞ=½Kmð1 þ I=KiÞg þ S ð1Þ

where viis the initial velocity at a given substrate

concen-tration with fluorogenic N-BK peptide; vothe initial velocity

at the same substrate concentration without fluorogenic

N-BK peptide; Kmthe Michaelis constant for the substrate;

Sthe chromogenic substrate (L-BAPA) concentration; I the

N-BK peptide concentration and the Kivalue corresponds

to the Michaelis constant for the N-BK peptide used as a

competitive substrate [31]

Identification of cleavage sites

Each protease (hK1, 17 nM; cathepsin B, 17 nM;

cathep-sin L, 1.7 n ; cathepsin K, 17 n ) was incubated with

N-BK peptide (17 lM) for 15 min at 37C in its respective assay buffer (final volume, 200 lL), and the reaction stopped by adding 800 lL ethanol The precipitate was removed and the supernatant, containing the native peptide and/or its proteolytic fragments, was evaporated to dryness, and redissolved in 0.1% trifluoroacetic acid An aliquot of each sample was fractionated by RP-chromatography on a

C18 Brownlee ODS-032 column, using a 35-min linear (0–60%) gradient of acetonitrile (in 0.1% trifluoroacetic acid) at a flow rate of 0.5 mLÆmin)1 Proteolysis products were identified by comparison with native peptidyl amides, and the elution profiles were analyzed using SPECTACLE software (ThermoQuest, les Ulis, France) [33] Cleavage sites were located by N-terminal sequencing (ABI 477 A sequencer, Applied Biosystems) The same experiments were carried out, varying incubation times from 15–60 min, with C-BK peptide (20 lM final), plus hK1 (2 nM) and cathepsins L (0.2 nM), K (2 nM) and B (2 nM)

Kallikrein hK1 (5 nM) and cathepsins L (0.5 nM), K (5 nM) and B (5 nM) were incubated with BK-peptide (52 lM) as above The kinins released were analysed by RP-HPLC (C18 ODS-032 column, 45-min linear (0–60%) gradient of acetonitrile (in 0.1% triluoroacetic acid), using

BK, BK(5–9), Lys-BK, and Des Arg9-BK for calibration The nature of the kinins released from BK-peptide were checked by N-terminal sequencing

Kininase activity of cathepsin K

BK, Lys-BK, and des-Arg9-BK were incubated with cath-epsin K for 0–120 min at 37C in 0.1Mphosphate buffer

pH 6.0, containing 2 mMdithiothreitol and 1 mMEDTA,

as above for the BK-peptide, and the products analysed by RP-chromatography (C18 Brownlee ODS-032 column, 45-min linear (0–60%) gradient of acetonitrile in 0.1% (TFA) Kinin metabolites were quantified by running the ChromQuest Chromatography Workstation (ThermoFin-nigan, les Ulis), and were identified by N-terminal peptide sequencing Similar experiments were performed with cathepsins B, L and hK1

Release of kinin from HMWK by cathepsins The release of kinin from kininogens by incubation with cathepsins B, L and K was measured by competitive enzyme immunoassay (Peninsula Laboratories, San Carlos,

CA, UK) Briefly, kininogens (final concentration, 2 nM) were incubated with increasing amounts of enzymes (kini-nogen/cathepsin molar ratio 1 : 4–10) in the assay buffer (final volume, 50 lL) at 37C for 0–240 min, and the reaction was stopped by adding ethanol [19] HMWK and T-kininogen were incubated similarly with trypsin and hK1, except that the buffer was 0.1M Tris/HCl buffer,

pH 8.5, 0.15M NaCl for trypsin, and 50 mM Tris/HCl buffer, pH 8.3, 1 mMEDTA for hK1 Kinins were further quantified by EIA, using biotinyl–bradykinin as tracer, and running theSOFTMAX PROsoftware (Thermomax microplate reader, Molecular Devices, Sunnyvale, CA, USA) The calibration curve was obtained by plotting the kinin centration against absorbance (450 nm) Under these con-ditions, bradykinin (BK), kallidin (Lys-BK), and [Tyr0]-BK were all 100% crossreactive, while [des-Arg9]-BK was not

detected The pH-dependent kininogenase activity of CP

was analyzed under similar experimental conditions, using

0.1Macetate buffer for pH 4–5, and 0.1MNaCl/Pifor pH

6–8

Results and discussion

Processing of HMWK by cathepsins

We reported previously that adding kininogen or cystatin to

cathepsins results in supplementary bands of digestion on

gelatin-containing SDS/PAGE, corresponding to protease–

inhibitor complexes [19] HMWK-bound cathepsins retain

some enzymatic activity towards peptide substrates when

they are incubated under stoichiometric conditions, as does

cathepsin L when bound to sheep stefin B [34] Although

the proteolytic activity of kininogen-bound enzyme was

stable and apparently unmodified by overnight incubation,

SDS/PAGE analysis indicated that cathepsin L generated

two major breakdown products from HMWK (Fig 1), as

observed for tissue kallikrein Cathepsins K and B

proc-essed HMWK similarly (not shown), as did the

trypano-somal CP, cruzipain [35] Accordingly, we observed the

presence of extralysosomal cathepsins B, L and K as active

forms in inflammatory bronchoalveolar lavage (BAL)

fluids, while kininogens were degraded; furthermore

addi-tion of intact HMWK to BAL fluid samples led to its rapid

and specific hydrolysis by CP (C Serveau, M Ferrer-Di

Martino, and G Lalmanach, unpublished observation)

Despite the fact that proteolysis of HMWK by CP may be

due to residual amounts of unbound cathepsin, the presence

of a reversible, covalent noninhibiting complex, as proposed

by Dennison et al [34], or the formation of an inappropriate

inhibitory complex [36,37] cannot be excluded

Enzymatic activity on fluorogenic kininogen-derived

peptides

We further analysed the kininogen processing using

kini-nogen-derived peptides, whose sequences are related to

human kininogens and surround residues Ile376 to Ile393

[38] (Fig 2A) Intramolecularly quenched fluorogenic

substrates (N-BK and C-BK peptides) were prepared as peptidyl-amides by Fmoc solid-phase synthesis, and were flanked by a fluorescent N-terminal Abz (ortho-aminoben-zoic acid) donor group and a C-terminal 3-NO2-Tyr (3-nitro-tyrosine) acceptor [39] Human tissue kallikrein (hK1), used as control, hydrolyzed the C-terminal derived peptide Abz-SPFRSSRI-(3-NO2-Tyr) more efficienly than Abz-ISLMKRPPGF-(3-NO2-Tyr) (Table 1) Although kininogen-derived substrates differ in length, their kcat/Km

Fig 1 High-molecular weight kininogen processing by hK1 and cathepsin L HMWK was incubated with cathepsin L or hK1 in their respective activity buffer (see the Experimental procedures section for details), and the products separated by SDS/PAGE on 10% gels under reducing conditions [28] Samples: lane 1, hK1; lane 2, hK1/HMWK (molar ratio, 0.1); lane 3, hK1/HMWK (molar ratio, 0.01); lane 4, HMWK; lane 5, cathepsin L/HMWK (molar ratio, 2); lane 6, cathepsin L/HMWK (molar ratio, 1); lane 7, cathepsin L/HMWK (molar ratio, 0.25); lane 8, cathepsin L.

Fig 2 Hydrolysis of kininogen-derived peptides and kinins by hK1 and cathepsins B, L and K (A) Structure of kininogen-derived fluorogenic substrates The sequence surrounding the region of bradykinin inser-tion corresponds to human kininogens [38] Bradykinin residues are shown in grey N-BK peptide, C-BK peptide and the BK-containing peptide (BK-peptide) were flanked by a donor-acceptor pair: a fluor-escent N-terminal Abz group and a C-terminal 3-NO 2 -Tyr quencher Peptides were synthesized as peptidyl-amides (B) N-BK peptide, C-BK peptide, BK-peptide and kinins (BK, Lys-BK, and des-Arg9-BK) were incubated with the enzyme, and samples were fractionated

by RP-HPLC (C18 Brownlee ODS-032 column; see Experimental procedures section for details), before the proteolysis products were identified by N-terminal peptide sequencing [32].

values compare with those reported previously [40,41].

Cathepsins K and L had high specificity constants towards

Abz-ISLMKRPPGF-(3-NO2-Tyr) (Table 1) While

cath-epsin L hydrolyzed the two fluorescent peptides similarly,

cathepsin K cleaved the substrate spanning the N-terminus

of bradykinin more efficiently Abz-SPFRSSRI-(3-NO2

-Tyr) was a rather poor substrate for human cathepsin B, but

this protease hydrolyzed Abz-ISLMKRPPGF-(3-NO2-Tyr)

with a kcat/Kmvalue similar to that of hK1 Cathepsins B, L

and K, and hK1 bound C-BK peptide with a higher affinity

than the N-BK peptide (Table 1) The Michaelis constants

for N-BK peptide were lower for cathepsins, and especially

for cathepsin L, than for hK1, but were identical for the

four enzymes towards C-BK peptide ( 1 lM) The similar

affinity pattern for all peptides indicates that the differences

in the variation of second-order rate constants are due

mostly to a change in the chemical reactivity (kcat)

Interestingly, hK1 looses its ability to hydrolyze the

kininogen-derived N-BK peptide after oxidization of

Met379 (i.e., two residues upstream of the N-terminus of

bradykinin) (kcat/Km< 2 mM )1Æs)1) as reported for

oxid-ized HMWK [13] On the other hand, cathepsin L remained

significantly active towards the oxidized N-BK peptide (kcat/

Km¼ 77 000M )1Æs)1), despite a decrease in the specificity

constant value Taking into account the abolition of kinin

release by kallikreins from oxidized kininogens [13], this

supports our initial hypothesis that cathepsin L may

represent an alternative, kallikrein-independent pathway in

the local kinin generation [19], despite the oxidizing

environment of the inflammatory focus

Both cathepsins, as well as hK1, hydrolyzed the C-BK

peptide [Abz-SPFRSSRI-(3-NO2-Tyr)] at the

Arg389-Ser390 bond (Fig 2B), as reported for the parent proteins

(i.e., LMWK and HMWK) under physiological conditions

No secondary cleavage site was identified Tissue kallikrein

cleaved N-BK peptide, i.e., Abz-ISLMKRPPGF-(3-NO2

-Tyr), at the Met379-Lys380 bond (kallidin-releasing site), as

did cathepsin K, in keeping with its preference for a leucyl

group at the S2 subsite (primary specificity pocket) [42] In

contrast, cathepsin L hydrolyzed the N-BK peptide mainly

at the Lys380-Arg381 bond (i.e., bradykinin-releasing site),

and to a lesser extent ( 20%) at the Met-Lys bond

(Fig 2B) The hydrolysis pattern of cathepsin B was clearly

different and related to its dicarboxypeptidase activity

[43,44] The enzyme cleaved the N-BK peptide at the

Gly-Phe bond, which led to the removal of the C-terminal Gly-

Phe-(3-NO2)-Tyr pair, reflecting its pronounced preference for

aromatic residues at P¢1 and P¢2 [45]

A longer substrate, encompassing human kininogen residues II(376–393) (BK-peptide, see Fig 2A), was used for further analysis of kininogen processing by CP For the sake of homogeneity with N-BK and C-BK peptides, the donor/acceptor pair was kept at the N- and C-terminal part

of BK-peptide This latter was rapidly cleaved by hK1, releasing kallidin, as for HMWK and LMWK under physiological conditions (Fig 2B) In agreement with the results reported above, bradykinin ( 80%) and kallidin were excised simultaneously upon incubation with cathep-sin L Under similar conditions, cathepcathep-sin B did not release any kinin from BK-peptide, and no hydrolysis products were detected, as reported for human and bovine kininogens [19] Incubation of BK-peptide with cathepsin K gave a different elution profile by RP-HPLC, no peak correspond-ing to commercial kinins used as standard However two specific cleavage sites were identified, one at the Gly384-Phe385 bond and the other at the Arg389-Ser390 bond, which is consistent with the unique ability of cathepsin K among mammalian cathepsins to accomodate Pro at P2 [46], resulting in the release of the 5-mer peptide, FSPFR(385–389), so called BK(5–9) Cathepsin L and hK1 cleaved Abz-ISLMKRPPGFSPFRSSRI-(3-NO2-Tyr) after Arg389, first releasing the kinin C-terminus, followed

by a second cleavage at the N-terminal part of BK This is similar to human plasma and porcine pancreatic kallikreins [13], and agrees with Km values, which indicated that cathepsin L and hK1 preferentially bind to the bradykinin C-terminus (Table 1) RP-HPLC analysis of the hydrolysis products also indicates that cathepsin K releases BK(5–9) via an initial cleavage of the Arg-Ser bond, followed by hydrolysis of the Gly-Phe bond (data not shown), but not via the initial production of kinin and the subsequent release

of a truncated fragment These data suggest that human cathepsin K proteolytically processes native kininogens, but, unlike cathepsin L, does not generate pharmacologi-cally active kinins

Kinin release from HMWK Cathepsins were incubated with HMWK, and the gener-ated kinins measured by ELISA, using an anti-bradykinin

Ig that reacted similarly with both Lys-BK and BK Cathepsin L liberated kinins, while cathepsins B and K did not generate immunoreactive kinins from HMWK (Fig 3) E-64 completely blocked the release of kinin by cathepsin L, while other class-specific low-molecular mass inhibitors had no effect While catalytic amounts of

Table 1 Hydrolysis of kininogen-derived fluorogenic substrates by hK1 and human cysteine proteases Second-order rate constants were measured under pseudo-first order conditions Kinetic data were determined by running the ENZFITTER software (Biosoft, Cambridge, UK), and were reported as means ± SD (triplicate experiments) Michaelis constants values were determined from Hanes linear plot, or under mixed alternative substrate conditions (34) as described in Material and methods Human tissue kallikrein was used as reference to calculate (k cat /K m )/(k cat /K m ) ref

N-BK peptide C-BK peptide

Enzyme k cat /K m (m M )1 Æs)1) k cat /K m )/(k cat /K m ) ref K m (l M ) k cat /K m (m M )1 Æs)1) (k cat /K m )/(k cat /K m ) ref K m (l M ) hK1 133 ± 5 1 10.9 ± 1.5 783 ± 11 1 0.9 ± 0.01 Cat L 5 850 ± 227 43.98 3.2 ± 0.05 4 428 ± 51 5.66 0.6 ± 0.03 Cat K 5 492 ± 468 41.28 7.1 ± 0.6 1 230 ± 28 1.57 1.7 ± 0.02 Cat B 331 ± 5 2.34 6.1 ± 0.04 33 ± 1 0.04 1.2 ± 0.03

cathepsin L hydrolyzed BK-peptide, kinin production

from HMWK required at least stoichiometric amounts

of CP In contrast, cathepsin L does not generate

immunoreactive kinins from rat T-kininogen (data not

shown), indicating that the release of kinin by cathepsin L

depends on its enzyme specificity Time-course

experi-ments with a HMWK/cathepsin L molar ratio of

1 : 0.25–4 showed that no detectable amount of

immu-noreactive kinins (BK) were released in less than 15 min

of incubation (minimum concentration, 1 pg per well)1,

i.e., 20 pgÆmL)1) The maximal kinin release (500 pgÆmL)1)

from HMWK was reached at t¼ 90 min, corresponding

to 80% of the total kinin content (610 pgÆmL)1of BK

eq per assay) Compared to the very rapid kinin release

by kallikreins, this slow production points to the

forma-tion of an inhibitory complex between cathepsin L with

HMWK (two cystatin-like inhibitory sites/molecule), and

demonstrates that the kininogenase activity occurs after

the partial hydrolysis of HMWK (Fig 1), as reported for

cystatin C-bound cathepsin L [37] The pH-dependent

kininogenase activity of cathepsin L over the pH range

4–8 gave a bell-shaped curve, showing that cathepsin L

liberated kinins optimally at pH 4.5 and 5 (Fig 4), in

agreement with its pH-dependent proteolytic activity

towards small peptide substrates

Kinin degradation by cathepsin K

The capacity of CP to metabolize kinins was further

analysed Cathepsin K catabolized kinins very rapidly and

efficiently, while tissue kallikrein and cathepsins L and B

did not Bradykinin was totally hydrolyzed in less than

5 min at an enzyme/substrate molar ratio of 1 : 10 000

(Fig 5) The hydrolysis product, BK(5–9), remained stable

after incubation for 2 h with active cathepsin K,

emphasi-zing the narrow kininase specificity of this enzyme BK,

Lys-BK and des-Arg9-Lys-BK were all cleaved by cathepsin K at the

Gly-Phe bond (Fig 2B), as was Abz-ISLMKRPPGFSP FRSSRI-(3-NO2-Tyr) Although the degradation of kinins

is mainly under the control of kininases, such as angioten-sin-converting enzyme (ACE) or carboxypeptidase N [2], other peptidases may be responsible for the breakdown of kinin at the site of inflammation It has been reported that p-chloromercuriphenylsulfonic acid, a thiol-specific inhib-itor, delays the breakdown of BK by macrophages more efficiently than does the carboxypeptidase inhitor,

D,Lmercaptomethyl-3-guanidino-ethylthiopropanoic acid [47], suggesting that an unindentified CP participates in the kinin degradation According to its great potency in catabolizing BK, Lys-BK and des-Arg9-BK in vitro, this CP from macrophages could be cathepsin K

In conclusion, the present report provides the first in vitro evidence that human cathepsin L may act as a kininoge-nase This could be of biological relevance at inflammatory

Fig 3 Release of immunoreactive kinins from human HMWK by

cathepsin L HMWK was incubated with cathepsins B, L and K

(0.1 M NaCl/P i , pH 6.0, 1 m M EDTA, 2 m M dithiothreitol for 60 min

at 37 C) with or without E-64 The amounts of kinin released

(expressed as BK eq.) were measured by competitive enzyme

immu-noassay, using biotinyl-bradykinin as the tracer [19] Kinin values were

normalized to the content of immunoreactive kinins generated by the

complete hydrolysis of human HMWK by trypsin According to the

antibody manufacturer, BK, Lys-BK and [Tyr0]-BK were all 100%

crossreactive, while [des-Arg9]-BK did not react with the

anti-bradykinin Ig.

Fig 4 pH-dependent kinin-releasing activity of cathepsin L HMWK was incubated with cathepsin L for 1 h at 37 C, using 0.1 M acetate buffer (pH 4–5) and 0.1 M NaCl/P i for pH 6–8 The kinin content (BK eq.) was measured by EIA, and the kinin values normalized as in Fig 3.

Fig 5 Kininase activity of cathepsin K Human cathepsin K was incubated with bradykinin (enzyme/substrate molar ratio, 1 : 10 000)

at 37 C, in 0.1 M NaCl/P i pH 6.0, containing 2 m M dithiothreitol and

1 m M EDTA for periods of 0–120 min Hydrolysis products were separated by RP-HPLC, using an analytical C 18 cartridge [45-min linear (0–60%) acetonitrile gradient in 0.1% trifluoroacetate] BK (black bar) and BK(5–9) (white bar) were quantified and normalized,

by running the chromatography workstation.

sites, where kinin production remains unaffected, although

kallikreins may loose their kininogenase properties [13]

Important amounts of CP are released from macrophages

during inflammation and colocalized with cystatins and

kininogens During the characterization of the proteolytic

activity of supernatants from inflammatory BAL fluids, we

have identified active forms of CPs (concentration in the

micromolar range), which mostly corresponds to

cathep-sin L, but also to cathepcathep-sins B, K, and -S, leading to a

disrupted CP/cystatin balance in favor of CP (estimated

ratio between 2–5 : 1, depending of the sample) (C Serveau,

M Ferrer-Di Martino, and G Lalmanach, unpublished

observation) This might allow cathepsin L to reach a

concentration level sufficient to match kinin production,

independently of kallikreins which are unable to generate

kinins under inflammatory conditions Furthermore, our

data indicate that cathepsin K is a highly potent

kinin-degrading enzyme that produces BK(5–9) from BK and

Lys-BK, and suggests that cathepsin K is a new member of

the kininase family Both the kininogenase activity of

cathepsin L and/or the kininase activity of cathepsin K may

be favored by the generation of an acidic environment in the

pericellular space around macrophages [20] Vasoactive

properties of human inflammatory BAL fluids are currently

under investigation using isolated bronchial tubes as a

model system Preliminary data support that our in vitro

findings are of physiological relevance (C Vandier, personal

communication)

Acknowledgements

We thank E Boll-Bataille´ for technical assistance and M

Brillard-Bourdet for N-terminal peptide sequencing The text was edited by

O Parkes This work was supported partly by an EU grant (Inco-Dev,

ICA4-CT2000-30035), by Biotechnocentre, and by a National Institute

of Health grant, AR46182 C D holds a doctoral fellowship from

MENRT (Ministe`re de l’Education Nationale, de la Recherche et de la

Technologie, France).

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