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 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:
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).
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