Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L Claire Desmazes 1 , Laurent Galineau 1 , Francis Gauthier 1 , Dieter Bro¨ mme 2 and Gilles Lalmanach 1 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; 2 Department 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 (k cat /K m ) 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, cathepsin 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) Eur. J. Biochem. 270, 171–178 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03382.x 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). N a -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 0.1 M phosphate buffer, pH 6.0 (containing 2 m M dithiothreitol and 1 m M EDTA). Enzymes were activated in their assay buffer for 5minat37°C 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 0.1 M Tris/HCl, pH 8.5, containing 0.15 M NaCl, and that for hK1 was 50 m M Tris/HCl, pH 8.3, containing 1 m M EDTA. 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 C 18 218TPS1 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 C 18 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 m M ) was incubated with aqueous N-chlorosuccinimide (ICN Pharmaceuticals, Orsay, France) (5 m M )in0.1 M Tris/HCl buffer, pH 8.5, for 1 h at room temperature to oxidize the methionyl group (Met379). The oxidized peptidewas purified by RP-HPLC (Vydac C 18 218TPS1 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 l M ) 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 0.1 M NaCl/P i ,pH6.0,1m M EDTA, 2 m M 172 C. Desmazes et al. (Eur. J. Biochem. 270) Ó FEBS 2003 dithiothreitol for 60 min at 37 °C. 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 m M Tris/HCl buffer, pH 8.3, EDTA 1 m M for 60 min at 37 °C. Kinetics measurement Determination of k cat /K m . 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 n M ),B (4 n M ), or L (2 n M )or hK1 (4 n M ) to N-BK peptide (final concentration: 0.5 l M ). Kinetic data were determined using the ENZFITTER software (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 n M )andL(6n M ). Determination of the Michaelis constant (K m ). K m values were determined from Hanes linear plots, with various concentrations of C-BK peptide (1–10 l M )pluscathep- sins L (6 n M ), K (4 n M )andB(3.7n M ), and hK1 (4 n M ). The Michaelis constants for hydrolysis of N-BK peptide by hK1 and cathepsin B were determined similarly, while the K m for 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 K m values for the fluorogenic substrate were obtained by measuring the dissociation constant (K i ) towards the chromogenic substrate. Assays were carried out by adding cathepsin L (60 n M )or cathepsin K (60 n M )toamixtureof L -BAPA (50–750 l M ) (whose K m are 86 l M and 66 l M , respectively) and N-BK peptide (1–10 l M ) [32]. The hydrolysis of L -BAPA was monitored at 410 nm (Hitachi U-2001 spectrophotometer), with less than 5% of L -BAPA hydrolyzed. The velocity of the reaction is described by: v i =v o ¼fðK m þ SÞ=½K m ð1 þ I=K i Þg þ S ð1Þ where v i is the initial velocity at a given substrate concen- tration with fluorogenic N-BK peptide; v o the initial velocity at the same substrate concentration without fluorogenic N-BK peptide; K m the Michaelis constant for the substrate; S the chromogenic substrate ( L -BAPA) concentration; I the N-BK peptide concentration and the K i value corresponds to the Michaelis constant for the N-BK peptide used as a competitive substrate [31]. Identification of cleavage sites Each protease (hK1, 17 n M ; cathepsin B, 17 n M ;cathep- sin L, 1.7 n M ; cathepsin K, 17 n M ) was incubated with N-BK peptide (17 l M )for15minat37°C 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 C 18 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 l M final), plus hK1 (2 n M )and cathepsins L (0.2 n M ), K (2 n M )andB(2n M ). Kallikrein hK1 (5 n M ) and cathepsins L (0.5 n M ), K (5 n M )andB(5n M ) were incubated with BK-peptide (52 l M ) 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 37 °Cin0.1 M phosphate buffer pH 6.0, containing 2 m M dithiothreitol and 1 m M EDTA, as above for the BK-peptide, and the products analysed by RP-chromatography (C 18 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 n M ) were incubated with increasing amounts of enzymes (kini- nogen/cathepsin molar ratio 1 : 4–10) in the assay buffer (final volume, 50 lL) at 37 °C 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 0.1 M Tris/HCl buffer, pH 8.5, 0.15 M NaCl for trypsin, and 50 m M Tris/HCl buffer, pH 8.3, 1 m M EDTA for hK1. Kinins were further quantified by EIA, using biotinyl–bradykinin as tracer, and running the SOFTMAX PRO software (Thermomax microplate reader, Molecular Devices, Sunnyvale, CA, USA). The calibration curve was obtained by plotting the kinin con- 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 Ó FEBS 2003 Cathepsin K: a new potent kininase (Eur. J. Biochem. 270) 173 detected. The pH-dependent kininogenase activity of CP was analyzed under similar experimental conditions, using 0.1 M acetate buffer for pH 4–5, and 0.1 M NaCl/P i for 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-NO 2 -Tyr (3-nitro-tyrosine) acceptor [39]. Human tissue kallikrein (hK1), used as control, hydrolyzed the C-terminal derived peptide Abz-SPFRSSRI-(3-NO 2 -Tyr) more efficienly than Abz-ISLMKRPPGF-(3-NO 2 -Tyr) (Table 1). Although kininogen-derived substrates differ in length, their k cat /K m 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]. 174 C. Desmazes et al. (Eur. J. Biochem. 270) Ó FEBS 2003 values compare with those reported previously [40,41]. Cathepsins K and L had high specificity constants towards Abz-ISLMKRPPGF-(3-NO 2 -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-NO 2 - Tyr) was a rather poor substrate for human cathepsin B, but this protease hydrolyzed Abz-ISLMKRPPGF-(3-NO 2 -Tyr) with a k cat /K m value 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 l M ). 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 (k cat ). 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) (k cat /K m <2m M )1 Æs )1 ) as reported for oxid- ized HMWK [13]. On the other hand, cathepsin L remained significantly active towards the oxidized N-BK peptide (k cat / K m ¼ 77 000 M )1 Æs )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-NO 2 -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-NO 2 - 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 Phe- (3-NO 2 )-Tyr pair, reflecting its pronounced preference for aromatic residues at P¢1andP¢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, cathepsin 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-NO 2 -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 K m 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 Ó FEBS 2003 Cathepsin K: a new potent kininase (Eur. J. Biochem. 270) 175 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 t ¼ 90 min, corresponding to  80% of the total kinin content (610 pgÆmL )1 of 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-BK were all cleaved by cathepsin K at the Gly-Phe bond (Fig. 2B), as was Abz-ISLMKRPPGFSP FRSSRI-(3-NO 2 -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 , L mercaptomethyl-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,thisCP 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 ,pH6.0,1m 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 1m 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 CHROMQUEST chromatography workstation. 176 C. Desmazes et al. (Eur. J. Biochem. 270) Ó FEBS 2003 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 cathepsins 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). References 1. Mu ¨ ller-Esterl, W. (1987) Novel functions of the kininogens. Semin. Thromb. Hemost. 13, 115–126. 2. Bhoola, K.D., Figueroa, C.D. & Worthy, K. (1992) Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol. Rev. 44, 1–80. 3. Regoli, D. & Barabe, J. (1988) Kinins receptors. Meth. Enzymol. 163, 210–230. 4. Duncan, A.M., Kladis, A., Jennings, G.L., Dart, A.M., Esler, M. & Campbell, D.J. (2000) Kinins in humans. Am. J. Physiol. 278, 897–904. 5. Emanueli, C. & Madeddu, P. (2001) Targeting kinin receptors for the treatment of tissue ischaemia. Trends Pharmacol. Sci. 22, 478–484. 6. Proud, D. & Kaplan, A.P. (1988) Kinin formation: mechanisms and role in inflammatory disorders. Annu. Rev. Immunol. 6, 49–83. 7. Blais, C., Jr, Marceau, F., Rouleau, J.L. & Adam, A. (2000) The kallikrein-kininogen-kinin system: lessons from the quantification of endogenous kinins. Peptides 21, 1903–1940. 8. Rocha e Silva, M., Beraldo, W.T. & Rosenfeld, G. (1949) Bra- dykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am. J. Physiol. 156, 261–273. 9. Owen, C.A. & Campbell, E.J. (1999) The cell biology of leukocyte- mediated proteolysis. J. Leukoc. Biol. 65, 137–150. 10. Jeffery, P.K. (1999) Differences and similarities between chronic obstructive pulmonary disease and asthma. Exp. Allergy 29, 14–26. 11. Taggart, C.C., Lowe, G.J., Greene, C.M., Mulgrew, A.T., O’Neill, S,J., Levine, R.L. & McElvaney, N.G. (2001) Cathepsin B, L, and S cleave and inactivate secretory leucoprotease inhibitor. J. Biol. Chem. 276, 33345–33352. 12. Wang, Z., Zheng, T., Zhu, Z., Homer, R.J., Riese, R.J., Chapman, H.A., Shapiro, S.D. & Elias, J.A. (2000) Interferon gamma induction of pulmonary emphysema in the adult murine lung. J. Exp. Med. 192, 1587–1600. 13.Kozik,A.,Moore,R.B.,Potempa,J.,Imamura,T.,Rapala- Kozik, M. & Travis, J. (1998) A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens. J. Biol. Chem. 273, 33224–33229. 14. Sato, F. & Nagasawa, S. (1988) Mechanism of kinin release from human low-molecular-mass-kininogen by the synergic action of human plasma kallikrein and leukocyte elastase. Biol. Chem. Hoppe Seyler 369, 1009–1017. 15. Ishii,Y.,Hashizume,Y.,Watanabe,T.,Waguri,S.,Sato,N., Ymamoto, M., Hasegawa, S., Kominami, E. & Uchiyama, Y. (1991) Cysteine proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung. J. Histochem. Cytochem. 39, 461–468. 16. Bu ¨ lhing, F., Reisenauer, A., Gerber, A., Kruger, S., Weber, E., Bro ¨ mme, D., Roessner, A., Ansorge, S., Welte, T. & Rocken, C. (2001) Cathepsin K – a marker of macrophage differentiation?. J. Pathol. 195, 375–382. 17. Chapman, H.A., Riese, R.J. & Shi, G.P. (1997) Emerging roles for cysteine proteases in human biology. Annu. Rev. Physiol. 59, 63–88. 18. Wolters, P.J. & Chapman, H.A. (2000) Importance of lysosomal cysteine proteases in lung disease. Respir. Res. 1, 170–177. 19. Desmazes, C., Gauthier, F. & Lalmanach, G. (2001) Cathepsin L, but not cathepsin B is a potential kininogenase. Biol. Chem. 382, 811–815. 20. Punturieri, A., Filippov, S., Allen, E., Caras, I., Murray, R., Reddy, V. & Weiss, S.J. (2000) Regulation of elastinolytic cysteine proteinase activity in normal and cathepsin K-deficient human macrophages. J. Exp. Med. 192, 789–799. 21. Henskens, Y.M., Veerman;, E.C. & Nieuw Amerongen, A.V. (1996) Cystatins in health and disease. Biol. Chem. Hoppe Seyler 2, 71–86. 22. Buttle, D.J., Abrahamson, M., Burnett, D., Mort, J.S., Barrett, A.J., Dando, P.M. & Hill, S.L. (1991) Human sputum cathepsin B degrades proteoglycan, is inhibited by alpha 2-macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B pre- cursor and cystatin C. Biochem. J. 276, 325–331. 23. Schmaier, A.H., Wahl, R., Fisher, S.J. & Brenner, D. (1998) The pharmacokinetics of the kininogens. Thromb. Res. 92, 293–297. 24. Linnevers, C.J., McGrath, M.E., Armstrong, R., Mistry, F.R., Barnes, M.G., Klaus, J.L., Palmer, J.T., Katz, B.A. & Bro ¨ mme, D. (1997) Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex. Protein. Sci. 6, 919–921. 25. Barrett, A.J., Kembhavi, A.A., Brown, M.A., Kirschke, H., Knight, C.G., Tamai, M. & Hanada, K. (1982) 1-trans-epoxy- succinyl-leucylamido (4-guanidino) butane (E-64) and its analo- gues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem. J. 201, 189–198. 26. Brillard-Bourdet, M., Moreau, T. & Gauthier, F. (1995) Substrate specificity of tissue kallikreins: importance of an extented inter- action site. Biochim. Biophys. Acta 1246, 47–52. Ó FEBS 2003 Cathepsin K: a new potent kininase (Eur. J. Biochem. 270) 177 27. Lalmanach, G., Adam, A., Moreau, T., Gutman, N.& Gauthier, F. (1991) Discrimination between rat thiostatin (T-kininogen) and one of its cystatin-like inhibitory fragments by a monoclonal anti- body, and localization of the epitope. Eur. J. Biochem. 196, 73–78. 28. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 29. Chagas, J.R., Authie, E., Serveau, C., Lalmanach, G., Juliano, L. & Gauthier, F. (1997) A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi. Mol. Biochem. Parasitol. 88, 85–94. 30. Serveau, C., Lalmanach, G., Juliano, M.A., Scharfstein, J., Juliano, L. & Gauthier, F. (1996) Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypa- nosoma cruzi, though the use of cystatin-derivated substrates and inhibitors. Biochem. J. 313, 951–956. 31. Segel, I.H. (1975) Enzyme Kinetics. John Wiley and Sons, New York. 32. Lecaille,F.,Authie,E.,Moreau,T.,Serveau,C.,Gauthier,F.& Lalmanach, G. (2001) Subsite specificity of trypanosomal cathe- psin 1-like cysteine proteases. Probing the S2 pocket with pheny- lalanine-derived amino acids. Eur. J. Biochem. 268, 2733–2741. 33. Lalmanach, G., Serveau, C., Brillard-Bourdet, M., Chagas, J.R., Mayer,R.,Juliano,L.&Gauthier,F.(1995)Conservedcystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases. J. Protein. Chem. 14, 645–653. 34. Pike, R.N., Coetzer, T.H.T. & Dennison, C. (1992) Proteolytically actve complexes of cathepsin L and a cysteine proteinase inhibitor; purification and demonstration of their formation in vitro. Arch. Biochem. Biophys. 294, 623–629. 35. Lima, A.P., Almeida, P.C., Tersariol, I.L., Schmitz, V., Schmaier, A.H., Juliano, L., Hirata, I.Y., Muller-Esterl, W., Chagas, J.R. & Scharfstein, J. (2002) heparan sulfate modulates kinin release by Trypanosoma cruzi through the activity of cruzipain. J. Biol. Chem. 277, 5875–5881. 36. Machleidt, W., Nagler, D.K., Assfalg-Machleidt, I., Stubbs, M.T., Fritz, H. & Auerswald, E.A. (1995) Temporary inhibition of papain by hairpin loop mutants of chicken cystatin. Distorted binding of the loops results in cleavage of the Gly (9)-Ala (10) bound. FEBS Lett. 361, 185–190. 37. Popovic, T., Cimerman, N., Dolenc, I., Ritonja, A. & Brzin, J. (1999) Cathepsin L is capable of truncating cystatin C of 11 N-terminal amino acids. FEBS Lett. 455, 92–96. 38. Takagaki, Y., Kitamura, N. & Nakanishi, S. (1985) Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens. J. Biol. Chem. 260, 8601–8609. 39. Meldal, M. & Breddam, K. (1991) Anthranilamide and nitrotyr- osine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal. Bio- chem. 195, 141–147. 40. Chagas, J.R., Portaro, F.C., Hirata, I.Y., Almeida, P.C., Juliano, M.A., Juliano, L. & Prado, E.S. (1995) Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallik- reins. Biochem. J. 306, 63–69. 41. Del Nery, E., Chagas, J.R., Juliano, M.A., Prado, E.S. & Juliano, L. (1995) Evaluation of the extent of the binding site in human tissue kallikrein by synthetic substrates with sequences of human kininogen fragments. Biochem. J. 312, 223–238. 42. Bro ¨ mme,D.,Klaus,J.L.,Okamoto,K.,Rasnick,D.&Palmer, J.T. (1996) Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsinO2incomparisonwithcathepsinsSandL.Biochem. J. 315, 85–89. 43. Illy, C., Quraishi, O., Wang, J., Purisima, E., Vernet, T. & Mort, J.S. (1997) Role of the occluding loop in cathepsin B activity. J. Biol. Chem. 272, 1197–1202. 44. Therrien, C., Lachance, P., Sulea, T., Purisima, E.O., Qi, H., Ziomek,E.,Alvarez-Hernandez,A.,Roush,W.R.&Menard,R. (2001) Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities. Biochemistry 40, 2702–2711. 45. Krupa, J.C., Hasnain, S., Na ¨ gler, D.K., Me ´ nard,R.&Mort,J.S. (2002) S2 substrate specificity and the role of His 110 and His 111 in the exopeptidase activity of human cathepsin B. Biochem. J. 361, 613–619. 46. Lecaille, F., Choe, Y., Brandt, W., Li, Z., Craik, C.S. & Bro ¨ mme, D. (2002) Selective inhibition in the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity. Biochemistry 41, 8447–8454. 47. Vietinghoff, G. & Paegelow, I. (2000) Degradation of bradykinin by peritoneal and alveolar macrophages of the guinea pig. Peptides 21, 1249–1255. 178 C. Desmazes et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . 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. 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. C-BK peptide (20 l M final), plus hK1 (2 n M )and cathepsins L (0.2 n M ), K (2 n M )andB(2n M ). Kallikrein hK1 (5 n M ) and cathepsins L (0.5 n M ), K (5 n M )andB(5n M ) were incubated with BK-peptide (52