Walls3, Lars Hellman4and Francis Gauthier1,2 1 Unite´ INSERM U-618 ‘Prote´ases et Vectorisation pulmonaires’, Tours, France 2 Universite´ Franc¸ois Rabelais de Tours, France 3 Immunophar
Trang 1G and chymase using fluorogenic substrates
Brice Korkmaz1,2, Gwenhael Je´got1,2, Laurie C Lau3, Michael Thorpe4, Elodie Pitois1,2, Luiz
Juliano5, Andrew F Walls3, Lars Hellman4and Francis Gauthier1,2
1 Unite´ INSERM U-618 ‘Prote´ases et Vectorisation pulmonaires’, Tours, France
2 Universite´ Franc¸ois Rabelais de Tours, France
3 Immunopharmacology Group, Sir Henry Wellcome Laboratories, Southampton General Hospital, UK
4 Department of Cell and Molecular Biology, The Biomedical Center, Uppsala University, Sweden
5 Departamento de Biofı´sica, Escola Paulista de Medicina, Universidade Federal, Sa˜o Paulo, Brazil
Introduction
Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC
3.4.21.39) are monomeric chymotrypsin-like serine
pro-teases that display a high degree of sequence similarity
and highly similar substrate specificity [1–3] They are
located predominantly in the primary granules of
neu-trophils and mast cells, respectively, although CG may
also be found in mast cells [4] The understanding of their distinctive roles in inflammatory events involving both neutrophils and mast cells can represent a chal-lenge as a result of their closely-related substrate specificities No substrate has been identified to date that allows differentiation of their activities when both
Keywords
cathepsin G; chymase; FRET substrate;
kinetics; mast cell; serine protease
Correspondence
B Korkmaz, Unite´ INSERM U-618
‘Prote´ases et Vectorisation pulmonaires’,
Universite´ Franc¸ois Rabelais de Tours,
37032 Tours, France
Fax: +33 2 47 36 60 46
Tel: +33 2 47 36 62 53
E-mail: brice.korkmaz@inserm.fr
(Received 4 April 2010, revised 11 May
2011, accepted 16 May 2011)
doi:10.1111/j.1742-4658.2011.08189.x
Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two clo-sely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and⁄ or neutrophils We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role dur-ing the progression of inflammatory diseases In addition to their prefer-ence for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1¢ subsite Despite significant structural differences in the S2¢ subsite, both prefer an acidic residue at that position The Ala226⁄ Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases However, a Lys at P1 is accommodated much less effi-ciently than a Phe, and the corresponding substrate is cleaved by b2-tryp-tase (EC 3.4.21.59) We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and b2-tryptase The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethy-lenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM )1Æs)1 This allows the quantification of active
CG in cells or tissue extracts where it may be present together with chym-ase and tryptchym-ase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma
Abbreviations
ABZ, O-aminobenzoic acid; ACT, antichymotrypsin; CG, cathepsin G; CMK, chloromethyl ketone; EDDnp,
N-(2,4-dinitrophenyl)-ethylenediamine; FRET, fluorescence resonance energy transfer; HNE, human neutrophil elastase; PR3, proteinase 3; Z, benzyloxycarbonyl.
Trang 2proteases are present Moreover, CG is weaker than
chymase at hydrolyzing most substrates currently
employed to quantify their activity and, accordingly,
this has hampered studies of their enzymatic properties
[4,5]
CG and chymase genes are located on chromosome
14 together with the genes of granzymes B and H [6]
The two proteases are synthesized as a prepro-protein,
containing a peptide signal, a prodipeptide and a
C-terminal propeptide [7] Mast cell chymase and CG
convert angiotensin I to the vasoactive peptide
angio-tensin II in human tissues [8], and this reaction may be
important in the progression to heart failure [9] and
aortic stenosis [10] Both proteases can also convert
the CXC chemokine connective tissue-activating
pep-tide III into active chemokine neutrophil-activating
peptide 2 through limited proteolysis [11], and both
are secretagogues for cultured serous cells [12] Mast
cell chymase and CG can also inactivate hepatocyte
growth factor [13] and both can degrade connective
tis-sue components such as fibronectin and vascular
endo-thelial cadherin [14] A close relationship between CG
and chymase is highlighted by the recent development
of a dual inhibitor, the administration of which has
been reported to be efficacious in the treatment of lung
inflammation in animal models [15]
The selective presence of CG in neutrophils confers
a destructive role on this protease with respect to the
degradation of pathogens within the phagolysosomes
[16] CG may also be secreted on neutrophil
activa-tion, and may remain associated with the neutrophil
membrane as an active protease [17] Soluble and
membrane-bound extracellular CG may participate in
the regulation of inflammatory processes through the
processing of chemokines⁄ cytokines and activation of
specific cell surface receptors [16,18] This protease
is also likely to contribute to the proteolysis of
con-nective tissue components in chronic inflammatory
disease [19]
Measuring protease-specific activities in situ is
criti-cal for the understanding of their distinctive functions,
as well as for the design of drugs that may be able to
regulate their activity Fluorescence resonance energy
transfer (FRET) substrates have proven to be valuable
alternatives to classical chromogenic and fluorogenic
substrates, both in terms of specificity and sensitivity
This is because FRET substrates allow an investigation
of protease specificity on both sides of the cleavage
site, unlike peptides with 4-nitroanilides, peptide
thiob-enzyl esters, 4-methyl-7-coumarylamide or
naphthyla-mides, which release chromophores or fluorophores
from the C-terminus [20,21] Moreover, FRET
substrates are particularly appropriate for a kinetic
investigation of neutrophil serine proteases because these proteases have an extended binding site on both the S and S¢ sides, as shown by X-ray analysis of the complex with inhibitors [22] Furthermore, synthesis of FRET substrates does not require sophisticated chemi-cal procedures and may be applied readily in the routine measurement of proteolytic activity in biologi-cal fluids or in fractionated cell suspensions [20] We and others have previously developed FRET substrates that are sufficiently sensitive to measure subnanomolar concentrations of human neutrophil elastase (HNE) (EC 3.4.21.37) and proteinase 3 (PR3) (EC 3.4.21.76) and CG [20,21,23] However, to date, no in depth investigation of the S¢ specificity of CG has been carried out that could aid the understanding of its pathophysiological function, and distinguish its activity from that of mast cell chymase Ultimately, a better knowledge of CG specificity should help in the devel-opment of a selective inhibitor of therapeutic interest
Results and Discussion
The crystal structure of CG in complex with the pept-idyl phosphonate inhibitor Suc-Val-Pro-PheP(OPh)2 exhibited the characteristic fold of chymotrypsin-like serine proteases and was very similar to that of human chymase [1] Preferential accommodation of a large hydrophobic residue in the S1 subsite of the two proteases is a result of the absence of a disulfide bond between Cys191 and Cys220, which is conserved in the neutrophil serine proteases HNE and PR3 The presence of a Glu at position 226 at the bottom of the
CG S1 subsite explains the accommodation of a posi-tively-charged P1 residue [1,24] Similar to other chymotrypsin-like serine proteases, CG and chymase preferentially accommodate a Pro at P2, and most of the commonly used chromogenic and fluorogenic substrates contain the Pro-Phe pair at P2–P1 [25,26]
A prolyl residue at the P2 position allows a change in the substrate chain as it threads through the active site, leading to an optimal positioning of the scissile bond
in the active site [25] Lys192 in CG and chymase has been suggested to favour interaction with a negatively-charged P3 residue [1] These observations explain the very similar substrate specificity of CG and chymase with both synthetic and natural substrates, although
CG generally cleaves synthetic substrates more slowly than do chymase and chymotrypsin-like proteases [5] The S¢ specificity of both CG and chymase is less well documented than S specificity; thus, a better knowl-edge of the combination between S and S¢ specificities could help to distinguish between the specificities of the two proteases
Trang 3S1¢ specificity of CG and chymase
The crystallographic data reported by Hof et al [1]
indicate that the side-chain of Arg41 located on the
30S insertion loop in CG projects from the molecular
surface to the east of the active site in accordance with
the standard orientation (Fig 1) Thus, the S1¢ pocket
in CG appears as a narrow crevice stabilized by the
Cys42-Cys58 disulfide bridge that defines the 30S loop
in both CG and chymase The S1¢ pocket is bordered
by His57 of the catalytic triad, Ser40 and Arg41,
whose flexibility allows it to be close to both the S1¢
and S2¢ subsites (Fig 1) Interestingly, an Arg residue
at position 41 is specific to CG and is shared only by
human and chimpanzee CG, suggesting a recent
appearance over the course of evolution (not shown)
In chymase, as in many other serine proteases, residue
41 is a Phe but, unlike other serine proteases, it
projects from the surface of the molecule and is
proxi-mal to the substrate P2¢ side-chain [27] Thus, the
chymase S1¢ pocket on the top of the Cys42-Cys58 loop is bordered by His57 to the west and the aliphatic part of the Lys40 side-chain to the east [2] (Fig 1) As
a result, the P1¢ specificity of chymase could be different from that of CG on account of the Lys40 in chymase helping to accomodate a negatively-charged P1¢ residue This could explain why chymase is more efficient than CG at inactivating bradykinin (RPPGFSPFRCOO)) upon cleavage of the C-terminal F–R bond [28] and it is likely that the Lys40 in chym-ase will form an electrostatic interaction with the negatively-charged carboxyl group of bradykinin To confirm this hypothesis, we raised two FRET sub-strates that had either an Asp or an Arg at P1¢ The peptidyl backbone of these substrates was that of a previously described FRET substrate: ABZ-GIA-TFCMLMPEQ-EDDnp (substrate 1) [where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophe-nyl)-ethylenediamine], which was derived from the inhibitory loop sequence of serpinB1 (previously called
Phe41
P1 P2
P3 P4
S1’
S2’
Arg143 Arg143
Lys217 Lys192
Lys192 Arg217
Ser40
P1 P2
P3
S2’
Cathepsin G
Chymase
Ser40
Arg143
Arg41 Lys192
Lys192
Phe41
His57 Ile99
Ile99 Cys42
Ser40
His57
Arg143 Lys40
S1’
A
B
S1’
Fig 1 Structural differences between CG
and chymase (A) The solvent accessible
surface based on the atom coordinates of
CG (1CGH) [1] and chymase (1PJP) [2] is
coloured to show positive (blue) and
nega-tive (red) electrostatic potentials The
irre-versible phosphonate inhibitors
Suc-Val-Pro-PheP-(OPh) 2 and
Suc-Ala-Ala-Pro-Phe-chlo-romethylketone complexed to CG and to
chymase, respectively, are shown as cyan
stick models The serine of the catalytic
triad is yellow (B) Ribbon plot of CG and
chymase in irreversible complexes with
syn-thetic inhibitors showing ball-and-stick
mod-els for the seven residues located in the
vicinity of the active site The molecular
sur-faces were generated using YASARA software
(http://www.yasara.org).
Trang 4monocyte neutrophil elastase inhibitor) and can be
cleaved at the F–C bond by CG and by chymase
[29,30] We found that the specificity constants,
kcat⁄ Km, for cleavage by CG and by chymase of
GIATFDMLMPEQ-EDDnp (substrate 2) and
ABZ-GIATFRMLMPEQ-EDDnp (substrate 3) were similar
in the 2· 102 mm)1Æs)1range (Table 1), indicating that
the Lys40 in chymase does not act as a discriminating
structural determinant of P1¢ specificity
The two best substrates developed previously for
CG, and which are also cleaved by chymase, differ
mainly in the size of the P1¢ residue One is derived
from the antichymotrypsin (ACT) sequence
(ABZ-TPFSGQ-EDDnp) and bears a Ser at P1¢ [31] and the
other, from a CG-cleaved sequence in
protease-acti-vated receptor-1, PAR-1 (ABZ-EPFWEDQ-EDDnp),
bears a Trp at this position [31,32] Because of the small
size of the S1¢ pocket in CG, we hypothesized that
small-sized residues are preferred by CG and that they
could possibly help to discriminate between CG and
chymase We introduced either a Ser or a Trp residue
at P1¢ in substrate 1 to obtain substrate 4
ATFSMLMPEQ-EDDnp) and substrate 5
(ABZ-GI-ATFWMLMPEQ-EDDnp) and tested these substrates
with CG and chymase As expected, cleavage sites
iden-tified by HPLC fractionation of the proteolysis
prod-ucts remained unchanged after the P1 Phe residue, and
a Trp residue at P1¢ significantly decreased the kcat⁄ Km; however, this result was obtained for both proteases (Table 1), which strongly suggests that S1¢ subsites in
CG and chymase are too closely related structurally to allow discrimination between these two proteases
S2¢ specificity of CG and chymase Crystallographic data show that the S2¢ subsite of CG
is highly polar as a result of the presence of three posi-tively-charged residues: Arg41, Arg143 and Lys192 [1] (Fig 1) In chymase, the Arg⁄ Phe substitution at posi-tion 41 projects the Phe side-chain into the active site cleft, resulting in partial obstruction at the bottom of the S2¢ subsite However, the crystal structure of chym-ase also indicates that the orientation of Arg143 in chymase differs from that in CG and is more proximal
to the S2¢ subsite This probably explains why, despite the Arg⁄ Phe substitution, chymase accomodates a neg-atively-charged P2¢ residue, as shown using a phage display random nonapeptide library (Fig 1) [33–35] Thus, CG and chymase could accommodate a negative P2¢ residue, although via a different mechanism that involves Arg41 and Lys192 in CG and Arg143 and Lys192 in chymase We have tested the influence of negative and positive residues at P2¢ in the serpinB1-derived FRET substrate to possibly take advantage of this different mechanism for discriminating between the two proteases We observed a significant increase
in specificity constant value using ABZ-GIATFCD-LMPEQ-EDDnp (substrate 6) compared to substrate 1 and a significant decrease in this rate constant using ABZ-GIATFCRLMPEQ-EDDnp (substrate 7) but, again, similar results were obtained with both chymase and CG Nevertheless, this demonstrates the impor-tance of the S2¢ subsite for both proteases, and also that Arg143 in chymase has a function similar to that
of Arg41 in CG (Table 1) This finding is in agreement with our observation that mouse CG, in which Arg41
is replaced by an Ala residue, cleaves substrates 6 and
7 at the same rate [24] Thus, despite the significantly different structure of their S2¢ subsite, CG and chym-ase have a similar preference for negatively-charged P2¢ residues We have previously shown that PR3 and HNE poorly accommodate a Pro at P2¢, which empha-sizes the importance of the S2¢ subsite in neutral serine proteases [36] Unlike PR3 and HNE, CG accommo-dates a P2¢ prolyl residue, as shown using substrate 8 (ABZ-GIATFCPLMPEQ-EDDnp), that is cleaved approximately twice as fast as control substrate 1 (Table 1) Again, however, the same result was obtained with chymase, further confirming the similar specificity of these two proteases
Table 1 Influence of residues at P1, P1¢ and P2¢ on the specificity
of CG and chymase as deduced from the specificity constant
kcat⁄ K m with FRET substrates derived from the serpinB1 and
ACT-reactive site loops Values (m M )1Æs)1) are the mean of‡ 3
experi-ments The error for kcat⁄ K m is < 15% The arrow indicates
cleav-age sites by CG and chymase NSH, no significant hydrolysis.
Number Substrates
kcat⁄ K m
Derived from SERPINB1
S1¢ specificity
S2¢ specificity
Derived from ACT
S1 specificity
a Value from Korkmaz et al [29] b Value from Re´hault et al [25].
Trang 5S1 specificity of CG and chymase
The dual specificity of CG for cleaving after large
hydrophobic or positively-charged residues has been
explained by the presence of a Glu residue at
position 226 at the bottom of the S1 pocket [1,24]
This idea has received support using mouse CG that
has an Ala at position 226 and does not cleave
P1-Lys containing substrates [24] and, more recently, as
a result of a phylogenetic analysis of mammalian
CGs [37] Human chymase also has an Ala residue
at position 226 and this could be exploited to raise
a specific CG substrate (Fig 2A) However, the
specificity constant for the reaction between CG and
a P1 Lys-containing substrate is far lower than
that of the corresponding substrate with a Phe at P1
[25]
The presence of an Ala residue at position 226 in
chymase also makes the S1 subsite wider, and this
could favour the accommodation of a P1 Trp residue
by chymase, as recently shown using a phage-displayed
selection of peptides susceptible to chymase cleavage
[34] We compared the hydrolysis by CG and chymase
of ABZ-TPFSALQ-EDDnp (substrate 9), ABZ-TP
KSALQ-EDDnp (substrate 10) and
ABZ-TPWSALQ-YNO2 (substrate 11) (Table 1) As expected, CG and
chymase prefered a Phe at P1 (substrate 9), although both also accommodated a Trp in their S1 subsite and only CG cleaved the P1Lys-containing substrate (Table 1) However, this occurred at a very low rate,
in accordance with previous findings [25] Because no other subsites from S2 to S3¢ in CG and chymase demonstrated a specificity that would allow discrimina-tion between the two proteases, we next attempted to improve the specificity constants of P1 Lys-containing substrates, aiming to measure subnanomolar amounts
of CG specifically
Design of specific and sensitive substrates for CG and chymase
A first step was to improve the kcat⁄ Km value of P1 Phe-containing substrates before substituting the P1-Phe by Lys Accordingly, we started from our most sensitive but not specific CG⁄ chymase FRET substrate ABZ-GIATFCDLMPEQ-EDDnp (substrate 6) and replaced the Thr residue by Pro [ABZ-GIAPFCDLM-PEQ-EDDnp (substrate 12)], aiming to prevent cleav-age at the C–D bond by HNE and PR3 with a Pro at P3 [38,39] and to improve cleavage by CG and chym-ase, although the latter prefers aliphatic residues at P2 [34] The Pro-Phe pair at P2–P1 is present in most of
Arg41
Asp147
Glu217
S3
Cathepsin G
P3
β2-Tryptase (monomer)
A
B
Fig 2 Structural differences between CG,
chymase and b2-tryptase (A) Ribbon plot of
CG and chymase in a complex with
syn-thetic inhibitors The irreversible
phospho-nate inhibitors Suc-Val-Pro-LysP-(OPh) 2 and
Suc-Ala-Ala-Pro-Phe-chloromethyl ketone in
a complex with cathepsin G and to
chym-ase, respectively, are shown as cyan stick
models Glu 226 and Ala 226 residues at the
bottom of the S1 subsite are shown in
green (B) Electrostatic surface potential of
human CG and b2-tryptase [50]
Solvent-accessible surfaces with a positive
electro-static potential are shown in dark blue, and
these with a negative electrostatic potential
are shown in red The serine of the catalytic
triad is shown in yellow The molecular
sur-faces were generated using using YASARA
software (http://www.yasara.org).
Trang 6the commonly used chromogenic and fluorogenic
substrates of CG that are also cleaved by chymase
[31] As expected, kcat⁄ Km of substrate 12 was
increased significantly using CG and chymase, and was
resistant to HNE cleavage (Table 2) However, this
substrate was still cleaved by PR3 at the C–D bond
(Table 2) Total resistance to PR3 hydrolysis was
obtained by substituting Ser for Cys in substrate 12 as
a result of the higher electronegative charge of the O
atom of the Ser side-chain compared to that of the
sulfur atom in the Cys side-chain; P3¢ Leu for Pro
because a Pro is not well accommodated by the PR3
S2¢ subsite [36]; and Ala for Glu at P3 because this
improves interaction with Lys192 at the S3 subsite of
CG The resulting substrate
(ABZ-GIEPFSDPMPEQ-EDDnp (substrate 13) fulfils most of the requirements
for CG, as well as for chymase cleavage (i.e a
nega-tively-charged residue at P3 and P2¢, a Pro-Phe pair at
P2–P1, and a Ser and a Pro at P1¢ and P3¢,
respec-tively), and this represents one of the most sensitive
substrates to have been reported for these two
prote-ases (Table 2) Finally, substituting Phe for Lys in this
optimized substrate [ABZ-GIEPKSDPMPEQ-EDDnp
(substrate 14)] totally abolished cleavage by chymase,
at the same time as maintaining specificity constant in
the 105m)1Æs)1 range (i.e sufficiently high to allow
specific measurements of nanomolar concentrations of
CG) (Table 2) As expected, HPLC analysis showed
that CG cleaved this substrate at the K–S bond
(Fig 3A) Furthermore, this substrate was not
hydro-lyzed by b2-tryptase (EC 3.4.21.59), despite the Lys at
P1 that is a preferential cleavage site for trypsin-like
proteases (Fig 3B) This was a result of the presence
of negatively-charged residues at P2 and P2¢ that are
not accommodated within the b2-tryptase active site
because of the presence of Asp147 and Asp143
within the S2 and the S2¢ subsites, respectively [40,41]
(Fig 2B) We cannot exclude the possibility,
how-ever, that trypsin-like protease(s) other than tryptase
are present in cells, tissues or biological fuids, such as
lung secretions and skin exudates, where CG and
chymase have been identified as critical pathophysio-logical actors Trypsin-like activities, however, could
be easily detected using broad spectrum inhibitors such as leupeptin or N-tosyl-l-lysine chloromethyl ke-tone that do not affect chymotrypsin-like proteases Nevertheless, we used a lysate of cells from a mast cell line and also sputum from a patient with severe asthma to measure hydrolysis of the newly-described substrate
Measurement of CG activity in a mast cell line extract and in sputum
Mast cells contain substantial amounts of a variety of proteases, including chymase, tryptase, carboxypepti-dase A3 and dipeptidyl pepticarboxypepti-dase I (cathepsin C), that participate in host defence and homeostasis [3] The qualitative and quantitative importance of CG or a CG-like protease in mast cells and mast cell lines remains unclear because the substrate specificity of
CG is close to that of chymase [42] and the corre-sponding mRNA has not been detected in the cell extracts [43] We used a mast cell line (HMC-1) extract
to measure CG activity using ABZ-GIEPKSDPM-PEQ-EDDnp (substrate 14) and evaluate its concentra-tion in comparison with that of chymase Accordingly,
we compared the rate of hydrolysis of the specific CG substrate and a CG⁄ chymase substrate by the cell extract, as well as by purified CG and chymase Opti-mized kinetic conditions were first determined to ensure that both substrates were cleaved at approxi-mately Vmax We measured CG activity in the HMC-1 cell line, which confirms previous results obtained using a specific trypsin-like fluorophosphonate probe [44] We ensured that the activity measured with ABZ-GIEPKSDPMPEQ-EDDnp was only a result of
CG by adding the irreversible chloromethylketone inhibitor Z-GLF-CMK (where Z is benzyloxycarbonyl and CMK is chloromethyl ketone), which specifically targets chymotrypsin-like proteases Full inhibition was obtained under these conditions, confirming
Table 2 Specificity constant k cat ⁄ K m for the hydrolysis of the FRET substrates derived from serpinB1 by CG, chymase, HNE and PR3 Values (m M )1Æs)1) are the means of‡ 3 experiments The error for k cat ⁄ K m is < 15% NSH, no significant hydrolysis.
Number Substrates derived from serpinB1
k cat ⁄ K m
Trang 7the specific role of CG in cleavage (Fig 4A) We
checked that this inhibitor did not alter cleavage by
the cell lysate of the trypsin-like substrate
ABZ-TPRSALQ-EDDnp at the R–S bond (not shown) We
also found that chymase activity was only twice as
high as that of CG in HMC-1 cells, in accordance
with preliminary observations made using MCTCmast
cells [4]
We also measured the hydrolysis of ABZ-GIEP
FSDPMPEQ-EDDnp (substrate 13) and ABZ-GIEP
KSDPMPEQ-EDDnp (substrate 14) by a sample of whole sputum from a patient with severe asthma Both substrates were rapidly cleaved at a single site identi-fied at the F-S bond and the K–S bond, respectively,
by HPLC analysis (Fig 4B) Cleavage was completely abolished after incubation with the chymotrypsin-like-specific Z-GLF-CMK inhibitor, which clearly demonstrates that no trypsin-like protease cleaved substrate 14 in the sputum (not shown) However, the resulting EDDnp-containing fragments from CG
ABZ-GIEPKSDPMPEQ-EDDnp
0 50 100 150 200 250
0 100 200 300 400 500 600
2000
1000
0
Elution time (min)
220 nm
320 nm
360 nm
SDPMPEQ-EDDnp
SDPMPEQ-EDDnp
ABZ-GIEPF
ABZ-GIEPK
Time (s)
ABZ-GIEPKSDPMPEQ-Y Cathepsin G+
ABZ-GIEPKSDPMPEQ-Y + Tryptase
ABZ-TPKSALQ-EDDnp + Tryptase
NO2
NO2
ABZ-GIEPFSDPMPEQ-EDDnp
A
B
Fig 3 Hydrolysis of
ABZ-GIEPFSDPMPEQ-EDDnp and ABZ-GIEPKSDPMPEQ-ABZ-GIEPFSDPMPEQ-EDDnp
by CG (A) Demonstration of identical
cleav-age sites within the two substrates as
visu-alized by reverse-phase HPLC and recording
at 360 nm of the EDDnp-containing
frag-ments (B) Control experiment showing no
cleavage of the Lys-containing CG substrate
ABZ-GIEPKSDPMPEQ-YNO2(20 l M ) by
b2-tryptase (10)7M final concentration) but
a rapid cleavage of ABZ-TPKSALQ-EDDnp
(20 l M ) by 10)9M b2-tryptase Hydrolysis of
ABZ-GIEPKSDPMPEQ-YNO2(20 l M ) by
10)9M CG is shown for comparison Assays
were carried out at 37 C in 50 m M Hepes
buffer (pH 7.4), 100 m M NaCl, 0.01% Igepal
CA-630 (v ⁄ v).
Trang 8hydrolysis (SDPMPEQ-EDDnp) were sequentially
degraded in a time-dependent manner This could be a
result of the presence of amino peptidase activity(ies)
in asthma sputum, although further work is required using larger numbers of sputum samples to confirm this hypothesis
Time (s)
Elution time (min)
ABZ-GIEPKSDPMPEQ-EDDnp
(substrate 14)
ABZ-GIEPKSDPMPEQ-EDDnp
(substrate 14)
ABZ-GIEPFSDPMPEQ-EDDnp
(substrate 13)
0
100 000
200 000
300 000
400 000
500 000
+ HMC-1 cells lysate
+ Asthma sputum
+ Asthma sputum
+ [HMC-1 cells lysate + Z-GLF-CMK]
Time (s)
0 400 800 1200 1600 2000
600 500 400 300 200 100
+ ABZ-GIEPFSY
Purified chymase
0
50
100
150
200
250
300
350
0
50
100
150
200
250
300
350
220 nm
320 nm
360 nm
SDPMPEQ-EDDnp
ABZ-GIEPF
ABZ-GIEPK B
A
SDPMPEQ-EDDnp
NO2
Fig 4 Hydrolysis of the CG substrate by a cell line extract and by a biological sample (A) Monitoring of ABZ-GIEPKSDPMPEQ-EDDnp hydrolysis by a HMC-1 mast cell lysate before and after incubation with the chymotrypsin-like protease inhibitor Z-GLF-CMK (3 m M final con-centration) The total inhibition observed in the presence of inhibitor indicates that the cleavage of the P1 Lys-containing substrate was a result of CG The insert shows the peptidase activity of purified chymase on a polyvalent substrate and its inability to cleave substrate 14 under the same experimental conditions (B) Hydrolysis of ABZ-GIEPFSDPMPEQ-EDDnp and ABZ-GIEPKSDPMPEQ-EDDnp by sputum from
a patient with severe asthma as visualized by reverse-phase HPLC and recording at 360 nm for the EDDnp-containing fragments Identical cleavage sites are observed within the two substrates but their cleavage was abolished after previous incubation with Z-GLF-CMK (not shown), indicating that only CG was involved in these cleavages Further degradation of the EDDnp-containing fragment, most probably by aminopeptidase activity present in the sputum, is observed for both peptides Assays were carried out at 37 C in 50 m M Hepes buffer (pH 7.4), 100 m M NaCl, 0.01% Igepal CA-630 (v ⁄ v).
Trang 9The reason why two closely-related proteases such
as chymase and CG are co-stored within the same cell
type remains unclear Mast cells are involved in a
variety of biological functions [45,46] and are mediated
by a range of potent mediators and proteases of
differ-ent specificities whose roles require clarification Using
a specific CG substrate such as that described in the
present study should help to define the roles of these
two proteases in diseases associated with mast cell
activation and facilitate the development of specific
inhibitors that could control their activity
Materials and methods
Materials
Purified CG (EC 3.4.21.20), HNE (EC 3.4.21.37) and ACT
were obtained from Biocentrum (Krakow, Poland) Purified
PR3 (EC 3.4.21.76) and b2-tryptase (EC 3.4.21.59) were
provided by Athens Research & Technology Inc (Athens,
GA, USA) and Merck (Nottingham, UK), respectively
Ige-pal CA-630 was obtained from Sigma (St Louis, MO,
USA) Z-GLF-CMK was obtained from Enzyme System
Products (Livermore, CA, USA) N,N-dimethylformamide
and acetonitrile were obtained from Merck (Darmstad,
Germany) Electrophoresis chemicals were obtained from
Bio-Rad (Marnes-la-Coquette, France) All other chemical
reagents were of analytical grade
Design and synthesis of quenched fluorescent
substrates
Quenched fluorogenic substrates were either obtained from
Genecust-Europe (Dudelange, Luxembourg) or prepared by
solid phase synthesis with Fmoc methodology [47]
Sub-strate purity was checked by MS (TofSpec-E; Micromass,
Manchester, UK) and by reversed-phase chromatography
on a C18 column The purified ABZ-peptidyl-EDDnp
con-centration was determined by measuring A365 with
e365= 17 300 m)1Æcm)1 for EDDnp [where ABZ is
O-am-inobenzoic acid and EDDnp is
N-(2,4-dinitrophenyl)-ethy-lenediamine] Stock substrate solutions (2–5 mm) were
prepared in 30% (v⁄ v) N,N-dimethylformamide and diluted
to 0.5 with 50 mm Hepes buffer (pH 7.4)
Enzyme assays
HNE, PR3 and CG were titrated with a1-proteinase
inhibi-tor, as described previously [48] Recombinant chymase,
produced and activated as described previously [34], was
titrated with ACT, the titre of which had been determined
by titration with CG Assays were carried out at 37C in
50 mm Hepes buffer (pH 7.4), 100 mm NaCl and 0.01%
Igepal CA-630 (v⁄ v) for CG; in 0.1 m Tris ⁄ HCl (pH 8.0)
and 50 mm Hepes (pH 7.4) for chymase; and in 750 mm NaCl and 0.05% Igepal CA-630 (v⁄ v) for HNE and PR3 The hydrolysis of ABZ-peptidyl-EDDnp substrates was monitored by measuring fluorescence at kex= 320 nm and
kex= 420 nm in a Hitachi F-2000 spectrofluorometer (Hit-achi, Tokyo, Japan) Specificity constants (kcat⁄ Km) were determined under first-order conditions, using a substrate concentration far below the estimated Kmas described pre-viously [31]
HMC-1 cells, kindly provided by Dr J H Butterfield (Mayo Clinic, Rochester, MN, USA) were cultured as described previously [42] Suspensions of 30–60 million cells were lysed in 2 mL of NaCl⁄ Pisupplemented with 1% Ige-pal CA-630 (v⁄ v) Proteolytic activity was measured at
37C using 50 lL of the cell lysates with ABZ-GIE-PFSDPMPEQ-EDDnp (25 lm) or ABZ-GIEPKSDPM-PEQ-EDDnp (25 lm) and 5 lL of cell lysate with ABZ-TPRSALQ-EDDnp (25 lm) in a total volume of
70 lL using a microplate fluorescence reader (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA, USA) under continuous stirring A sample of induced sputum from a patient with severe asthma was kindly provided by Dr Peter
H Howarth (University of Southampton, Southampton, UK) Written informed consent was obtained from the patient from whom the sputum sample was obtained
Chromatographic procedures and analysis of peptide products
Once the enzyme–substrate reaction was complete, the reac-tion medium was incubated with four volumes of absolute ethanol for 15 min on ice and centrifuged at 13 000 g for
10 min The supernatant containing the hydrolysis products was recovered, air-dried under vacuum and dissolved in
200 lL of 0.0075% trifluoroacetic acid (v⁄ v) Hydrolysis fragments were fractionated by reversed-phase HPLC and eluted peaks were monitored at three wavelengths (220, 320 and 360 nm) simultaneously, which allowed direct identifi-cation of EDDnp-containing peptides before sequencing or
MS analysis to identify cleavage sites
Nomenclature The nomenclature used for the individual amino acid resi-dues (e.g P2, P1, P1¢, P2¢, etc.) of a substrate and corre-sponding residues of the enzyme subsites (e.g S2, S1, S1¢, S2¢, etc.) follows that of Schechter and Berger [49]
Acknowledgements
This work was supported by ‘Region Centre’ and the
‘Fonds Europe´en de De´veloppement Re´gional’ (Projet INFINHI) and Agence Nationale pour la Recherche (project ANR-07-PHYSIO-029-01) The authors thank
Trang 10Miche`le Brillard-Bourdet for sequence analyses;
Chris-tophe Epinette and Lise Vanderlynden for technical
support; Dr Peter H Howarth, University of
South-ampton, for providing a sputum sample; and the
‘Plate-forme d’Analyse Inte´grative des Biomarqueurs’
for MALDI-TOF MS analyses
References
1 Hof P, Mayr I, Huber R, Korzus E, Potempa J, Travis
J, Powers JC & Bode W (1996) The 1.8 A crystal
structure of human cathepsin G in complex with
Suc-Val-Pro-PheP-(OPh)2: a Janus-faced proteinase with
two opposite specificities EMBO J 15, 5481–5491
2 Pereira PJ, Wang ZM, Rubin H, Huber R, Bode W,
Schechter NM & Strobl S (1999) The 2.2 A crystal
structure of human chymase in complex with
succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural
expla-nation for its dipeptidyl carboxypeptidase specificity
J Mol Biol 286, 163–173
3 Trivedi NN & Caughey GH (2010) Mast cell peptidases:
chameleons of innate immunity and host defense Am
J Respir Cell Mol Biol 42, 257–267
4 Schechter NM, Irani AM, Sprows JL, Abernethy J,
Wintroub B & Schwartz LB (1990) Identification of a
cathepsin G-like proteinase in the MCTC type of
human mast cell J Immunol 145, 2652–2661
5 Schechter NM, Choi JK, Slavin DA, Deresienski DT,
Sayama S, Dong G, Lavker RM, Proud D & Lazarus
GS (1986) Identification of a chymotrypsin-like
protein-ase in human mast cells J Immunol 137, 962–970
6 Fellows E, Gil-Parrado S, Jenne DE & Kurschus FC
(2007) Natural killer cell-derived human granzyme H
induces an alternative, caspase-independent cell-death
program Blood 110, 544–552
7 Salvesen G, Farley D, Shuman J, Przybyla A, Reilly
C & Travis J (1987) Molecular cloning of human
cathepsin G: structural similarity to mast cell and
cytotoxic T lymphocyte proteinases Biochemistry 26,
2289–2293
8 Reilly CF, Tewksbury DA, Schechter NM & Travis J
(1982) Rapid conversion of angiotensin I to angiotensin
II by neutrophil and mast cell proteinases J Biol Chem
257, 8619–8622
9 Jahanyar J, Youker KA, Loebe M, Assad-Kottner C,
Koerner MM, Torre-Amione G & Noon GP (2007)
Mast cell-derived cathepsin g: a possible role in the
adverse remodeling of the failing human heart J Surg
Res 140, 199–203
10 Helske S, Syvaranta S, Kupari M, Lappalainen J, Laine
M, Lommi J, Turto H, Mayranpaa M, Werkkala K,
Kovanen PT et al (2006) Possible role for mast
cell-derived cathepsin G in the adverse remodelling of
ste-notic aortic valves Eur Heart J 27, 1495–1504
11 Schiemann F, Grimm TA, Hoch J, Gross R, Lindner B, Petersen F, Bulfone-Paus S & Brandt E (2006) Mast cells and neutrophils proteolytically activate chemokine precursor CTAP-III and are subject to counterregula-tion by PF-4 through inhibicounterregula-tion of chymase and cathep-sin G Blood 107, 2234–2242
12 Sommerhoff CP, Nadel JA, Basbaum CB & Caughey
GH (1990) Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells
J Clin Invest 85, 682–689
13 Raymond WW, Cruz AC & Caughey GH (2006) Mast cell and neutrophil peptidases attack an inactivation segment in hepatocyte growth factor to generate NK4-like antagonists J Biol Chem 281, 1489–1494
14 Mayranpaa MI, Heikkila HM, Lindstedt KA, Walls
AF & Kovanen PT (2006) Desquamation of human coronary artery endothelium by human mast cell prote-ases: implications for plaque erosion Coron Artery Dis
17, 611–621
15 Maryanoff BE, de Garavilla L, Greco MN, Haertlein
BJ, Wells GI, Andrade-Gordon P & Abraham WM (2009) Dual inhibition of cathepsin G and chymase is effective in animal models of pulmonary inflammation
Am J Respir Crit Care Med 181, 247–253
16 Pham CT (2006) Neutrophil serine proteases: specific regulators of inflammation Nat Rev Immunol 6, 541– 550
17 Owen CA & Campbell EJ (1999) The cell biology
of leukocyte-mediated proteolysis J Leukoc Biol 65, 137–150
18 Meyer-Hoffert U (2009) Neutrophil-derived serine pro-teases modulate innate immune responses Front Biosci
14, 3409–3418
19 Korkmaz B, Horwitz M, Jenne DE & Gauthier A (2010) Neutrophil elastase, proteinase 3 and cathepsin
G as therapeutic targets in human diseases Pharmacol Rev 62, 726–759
20 Carmona AK, Juliano MA & Juliano L (2009) The use
of fluorescence resonance energy transfer (FRET) pep-tides for measurement of clinically important proteolytic enzymes An Acad Bras Cienc 81, 381–392
21 Korkmaz B, Attucci S, Juliano MA, Kalupov T, Jourdan
ML, Juliano L & Gauthier F (2008) Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates Nat Protoc 3, 991–1000
22 Korkmaz B, Moreau T & Gauthier F (2008) Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions Biochimie 90, 227–242
23 Lesner A, Wysocka M, Guzow K, Wiczk W, Legowska
A & Rolka K (2008) Development of sensitive cathepsin
G fluorogenic substrate using combinatorial chemistry methods Anal Biochem 375, 306–312