Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples

Article abstract of DOI:10.1016/j.abb.2016.10.007

Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S′ specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO₂)-NH₂, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.

Full text of DOI:10.1016/j.abb.2016.10.007

Accepted Manuscript
Development of the first internally-quenched fluorescent substrates of human
cathepsin C: The application in the enzyme detection in biological samples
Monika Łęgowska, Yveline Hamon, Anna Wojtysiak, Renata Grzywa, Marcin
Sieńczyk, Timo Burster, Brice Korkmaz, Adam Lesner
PII:
S0003-9861(16)30397-6
10.1016/j.abb.2016.10.007
YABBI 7387
DOI:
Reference:
To appear in: Archives of Biochemistry and Biophysics
Received Date: 31 August 2016
Accepted Date: 11 October 2016
Please cite this article as: M. Łęgowska, Y. Hamon, A. Wojtysiak, R. Grzywa, M. Sieńczyk, T. Burster,
B. Korkmaz, A. Lesner, Development of the first internally-quenched fluorescent substrates of human
cathepsin C: The application in the enzyme detection in biological samples, Archives of Biochemistry
and Biophysics (2016), doi: 10.1016/j.abb.2016.10.007.
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ACCEPTED MANUSCRIPT
Development of the first internally-quenched fluorescent substrates of human cathepsin
C: the application in the enzyme detection in biological samples
Monika ŁĘGOWSKA*, Yveline HAMON†, Anna Wojtysiak*, Renata GRZYWA‡, Marcin
1
SIEŃCZYK‡, Timo BURSTER§, Brice KORKMAZ†, Adam LESNER*
*
†
Faculty of Chemistry, University of Gdansk, 80-308 Gdansk, Poland
INSERM U-1100, “Centre d’Etudes des Pathologies Respiratoires” and Université François
Rabelais, 37032, Tours, France
‡
Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland
Department of Neurosurgery, Ulm University Medical Centre, 89081 Ulm, Germany
to whom correspondence should be addressed (email: adam.lesner@ug.edu.pl)
§
1
Short title: FRET substrates of human cathepsin C

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Abstract
Cathepsin C is a wildly expressed cysteine exopeptidase that is mostly recognized for the
activation of the granule-associated proinflammatory serine proteases in neutrophils,
cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted
extracellularly; however, its occurrence in human bodily fluids/ physiological samples has not
been thoroughly studied. In the course of this study, the first fluorescence resonance energy
transfer peptides for the measurement of the activity of human cathepsin C were designed and
synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S′
specificity study of cathepsin C, which has been examined for the first time. The extensive
enzymatic studies of the obtained compounds resulted in the selection of the highly specific
and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH , which was successfully
2
2
employed for the detection of cathepsin C activity in complex biological samples such as cell
lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate
was performed in order to better understand the binding mode of the substrates in the active
site of cathepsin C.
Keywords: FRET substrates, cysteine proteases, cathepsin C, peptidomimetics
Abbreviations used: ABZ, 2-aminobenzoic acid; ACC, 7-aminocoumarin-4-acetic acid;
AMC, 7-amino-4-methylcoumarin; Ala(Mca), β-(7-methoxy-coumarin-4-yl)-alanine; BALF,
bronchoalveolar lavage fluid; Bip, biphenylalanine; Cat, cathepsin; CCA, α-cyano-4-hydroxy-
cinnamic acid; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; DHB, 2,5-
dihydroxybenzoic; DIPCI, N,N′-diisopropylcarbodiimide; DIP, desquamative interstitial
pneumopathy; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; E64,
trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; HBTU, N,N,N′,N′-tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; HNE,
human neutrophil elastase; HX, histiocytosis X; Nle(6-OBzl), 6-benzyloxynorleucine; NSPs,
neutrophil serine proteases; PR3, proteinase 3; Thi, β-(2-thienyl)-alanine; Thi-Phe-CN, β-(2-
thienyl)-alanylphenylalanine nitrile; TFA, trifluoroacetic acid;

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INTRODUCTION
Cysteine cathepsins are ubiquitously expressed lysosomal proteases, initially considered to be
responsible exclusively for protein degradation during necrotic and autophagic cell death.
Over the last 60 years since the discovery of lysosomes, cathepsins have been proven to
display highly specific and strict proteolytic activity in various important physiological
processes, including protein activation, antigen processing or extracellular matrix remodeling
during wound healing and many others (1) (2). Cathpesin C (Cat C, also known as dipeptidyl
peptidase I, EC 3.4.14.1) due to its oligomeric structure, exodipeptidase activity and intricate
activation mechanism is a unique member of cysteine proteases (3) (4). Cat C exists as a
homotetramer of 200 kDa and each monomer is composed of three tightly joined peptide
chains. It contains a heavy and a light chain, which form the catalytic domain adopting the
papain-like structure, common for all cysteine cathepsin endopeptidases, and additionally
atypical N-terminal exclusion domain (3). The exclusion domain blocks the enzyme binding
site beyond the S pocket (nomenclature according to the Schechter-Berger model (5)), thus is
2
responsible for the ability of Cat C to remove dipeptides from the N-terminus of the substrate.
The enzyme cleaves two-residue units until it reaches a stop sequence: a basic side chain
residue (Arg, Lys, Orn) in P position, isoleucine residue in P position or proline residue
2
1
either in P or P ′ position (6). Due to dipeptidase activity, most of the reported synthetic
1
1
cathepsin C substrates are dipeptides bearing various reporter groups. Over the years, glycyl-
-phenylalanyl-7-amino-4-methyl-coumarin (Gly-Phe-AMC) has become the most commonly
L
used fluorogenic substrate, however, many synthetic dipeptide-based AMC substrates have
been used to characterize the broad specificity of cathepsin C (6) (7). The optimal substrates
contain small aliphatic residues such as Ala, Abu or Met in P position. The P position is
2
1
usually occupied by hydrophobic amino acid residues such as Phe, homophenylalanine (Hph)
or 6-benzyloxynorleucine (Nle(6-OBzl)), nevertheless Arg is also well tolerated at this
position. A number of different assay formats have also been reported to measure intracellular
activity of human cathepsin C including activity-based probes (8) (9) and rhodamine
substrate, (H N-Abu-Hph) -Rd, suitable for FACS (8).
2
2
The mostly recognized physiological substrates of Cat C are granule-associated serine
proteases: human neutrophil elastase (HNE), cathepsin G (Cat G), protainase 3 (PR3) and
neutrophil serine proteinase 4 (NSP4) from neutrophils, mast cell chymase, as well as
granzymes A and B from cytotoxic T lymphocytes and natural killer cells (10) (11) (12) (13).
Cat C was also found to participate in neuraminidase activation (14), platelet factor XIII

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activation (15) and regulation of plasminogen-plasmin system (16). It has been shown that
mutations in human Cat C that lead to loss of its function result in a decrease in host defense
and increased susceptibility to microbe infection being primarily responsible for Papillon-
Lefevre (17) and Haim-Munk syndromes development (18). The studies using mice models
showed that Cat C deficiency renders the resistance to develop sepsis (19), arthritis (20) and
abdominal aortic aneurysms (21). Therapeutic Cat C inhibitors should prevent the conversion
of proforms of neutrophil serine proteases into active enzymes so that neutrophils are unable
to accumulate at the site of inflammation due to the impaired chemotaxis (22). Thus, human
Cat C has been considered a promising drug target for the treatment of chronic inflammatory
diseases (22) (23). Very recently, we have investigated the occurrence of Cat C in
hematopoietic precursor cells, as well as in differentiated mature immune cells (neutrophils,
alveolar macrophages) in lungs and in lung airway fluids (24). We have shown that active Cat
C is present at high level in sputa from patients with lung inflammation, after being secreted
by activated neutrophils, thus, active Cat C may serve as a sensitive marker of neutrophilic
inflammation. Pathophysiological role of Cat C has been rarely studied in the past, so a
selective/sensitive detection of the enzyme activity is significant for a deeper insight into its
molecular function and its diagnostic potential.
The aim of presented study was the synthesis of novel, highly specific and selective human
Cat C substrates, suitable for the detection of its proteolytic activity in complex biological
samples. In order to achieve these goals, two series, each of 19 intramolecular fluorescence-
quenched substrates were synthesized and studied. Firstly, the suitable donor and acceptor for
fluorescence resonance energy transfer were selected, considering their optimal positions
within the peptide chain. Next, based on the chosen donor-acceptor pair, the length of the
peptide chain was extended in order to increase the specificity of the compounds by
expanding interactions with Cat C primed subsites to gain an insight into the S′ specificity of
the enzyme, primarily S ′ and S ′ binding pockets. Such an approach to map the S′ subsites of
1
2
Cat C has never been done and no FRET substrates have been reported so far for this
aminodipeptidase. Finally, we have evaluated the utilization of obtained compounds for the
measurement of Cat C activity in complex biological samples of different origin.

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EXPERIMENTAL
Chemical synthesis
Chemical synthesis of fluorogenic dipeptide substrates
-amino-4-methylcoumarin (AMC) was purchased from Sigma Aldrich (USA). 7-Fmoc-
7
aminocoumarin-4-acetic acid (Fmoc-ACC) was obtained as reported before (25). Boc-β-(2-
thienyl)-alanine (Boc-Thi) was purchased from Bachem (Switzerland).
The synthesis of Gly-Phe-ACC and Thi-Phe-ACC was performed on solid support using
Fmoc/tBu strategy on the Tenta Gel S RAM resin. The Fmoc-ACC was attached to the resin
using the procedure described before (25). Fmoc deprotection was performed with 20%
piperidine in dimethylformamide (DMF), whereas the chain elongation was achieved with
standard DIPCI (N,N′-diisopropylcarbodiimide)/HOBt (N-hydroxybenzotriazole) chemistry
using 3 molar eq. of protected amino acid derivatives. After completing the synthesis, the
final products were cleaved off the resin using TFA/H O/phenol/triisopropylsilane (88:5:5:2,
2
v/v/v/v) mixture.
The synthesis of the AMC substrates was performed in solution using Boc strategy. Briefly,
AMC was coupled to Boc-Phe-OH followed by the removal of Boc group and subsequent
coupling either with Boc-Gly-OH or Boc-Thi-OH. The peptide synthesis was performed in
acetonitrile using HBTU as the coupling agent in the presence of diisopropylethylamine
(DIPEA). Finally, the Boc group was removed with 50% TFA in dichloromethane (DCM).
The progress of the reactions was monitored by TLC.
Chemical synthesis of FRET substrates
Fmoc-β-(7-methoxy-coumarin-4-yl)-Ala (Fmoc-Ala(Mca)) was purchased from Bachem. The
FRET peptides were synthesized manually via the solid-phase method using the Fmoc/tBu
approach on Tenta Gel S RAM resin (substitution 0.24 meq/g; RAPP Polymere, Germany).
Peptide chains were elongated in cycles of deprotection and coupling steps using 20%
piperidine in DMF for the removal of Fmoc group and standard DIPCI/HOBt coupling system
for the amide bond formation using 3 molar excess of protected amino acid derivative for
each step. Finally, Boc-Thi-OH was conjugated as the N-terminal residue followed by a
complete peptide deprotection and simultaneous cleavage off the resin using a
TFA/H O/phenol/triisopropylsilane (88:5:5:2, v/v/v/v) mixture.
2

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Chemical synthesis of Thi-Ala(Mca)
The Fmoc group of Fmoc-Ala(Mca) was removed with 1,8-diazabicyclo[5.4.0]undec-7-ene
DBU) in tetrahydrofuran using N-(2-mercaptoethyl)aminomethyl polystyrene as a
(
dibenzofulvene scavenger (26). Next, the Ala(Mca)-OMe was synthesized using trimethylsilyl
chloride (TMSCl) in methanol (27) followed by Boc-Thi conjugation in acetonitrile using
HBTU in the presence of DIPEA as the coupling agent. After the reaction was completed, the
standard ethyl acetate work-up was performed. Finally, Boc group was removed with 50%
TFA in DCM.
MALDI-TOF mass spectrometry analysis was performed to identify all synthesized
compounds (Biflex III MALDI–TOF mass spectrometer using the α-cyano-4-hydroxy-
cinnamic acid (CCA) or 2,5-dihydroxybenzoic acid (DHB) matrix). The purity of the final
products was analyzed by RP-HPLC on a Pro Star system (Varian, Australia) equipped with a
Gemini 5µm C18 110Å (150x4.6 mm) column (Phenomenex, USA) and UV–VIS detector.
Enzymatic studies
Recombinant human proCat C and Cat L were purchased from R&D Systems (USA) and used
for the initial substrate activity studies. Recombinant human Cat C used for the specificity
study was kindly provided by John Pedersen from Unizyme Laboratories (Hørsholm,
Denmark). Cat B from human liver was obtained from Calbiochem (USA). Papain from
papaya latex and E64 were purchased from Sigma Aldrich (USA). Z-Phe-Arg-AMC was from
Bachem (Switzerland). Human cystatin C was a kind gift from Izabela Behrendt (University
of Gdansk, Poland).
Measurements of cathepsin C and cathepsin L activity
Recombinant human Cat L (R&D Systems, USA) was used to activate Cat C following
manufacturer recommendations. The substrates were incubated in a 96-well white microtiter
plate at a volume of 200 µl and concentration of 7.5 µM in 25 mM MES buffer containing 50
mM NaCl and 2 mM DTT, pH 6.0, at 37 °C. The enzymes were added at the concentration
ranging from 0.05 nM to 5 nM. The change of fluorescence intensity was monitored over
time. All measurements were carried out in triplicate and recorded on a FluoroSTAR
microplate reader (BMG, Germany) equipped with a set of 12 extinction emission filters.
Coumarins AMC and ACC were excited at 360 nm and their emission was followed at 450
nm, whereas Ala(Mca) was excited at 320 nm and its emission recorded at 400 nm.

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Substrate kinetics
In order to determine the kinetic parameters (K , k and k /K ) for the selected substrates
M
cat
cat
M
with Cat C and Cat B, the buffered solution of the enzyme was added to substrate solution at
concentration ranging from 0.3 to 40 µM or from 0.3 to 75 µM, for Cat C and Cat B,
respectively. Two buffers were used: sodium acetate buffer at pH 5 (50 mM CH COONa, 50
3
mM NaCl, 2 mM DTT, 1 mM EDTA) and MES at pH 6 (as described above). The change of
fluorescence intensity was observed over time. The titration curve for both AMC and Thi-
Ala(Mca) was obtained by measuring the fluorescence change as a function of concentration
and the data were used to calculate the concentration of the donor and the rate of substrate
hydrolysis. The initial hydrolysis rates were measured in triplicate and each experiment was
performed at least in duplicate. The determined constant values are given as an average with
standard deviation.
Cat C was used at concentration of 100 to 130 pM (for Thi-Phe-AMC) and 64 to 105 pM (for
FRET substrates), calculated for the tetramer content. The active Cat C concentration was
determined similarly as described by Tran et al. (6). Briefly, the active site concentration of
papain was determined by titration with E-64 and then was used to determine the
concentration of human cystatin C. The papain activity was measured using Z-Phe-Arg-AMC
(
λ =360 nm and λ =450 nm) in 50 mM HEPES, 2.5 mM DTT and 2.5 mM EDTA, pH 7.5.
ex em
Finally, human cystatin C was used for the titration of activated cathepsin C, which was found
to be 40-50% active. Cat B was titrated with E64 as described above for papain and used for
kinetic studies at 1 nM concentration.
Specificity study
Recombinant human Cat C (crystallization grade) from Unizyme Laboratories was used to
study the enzyme specificity at different pH conditions. Three buffer systems were applied:
sodium acetate at pH 5 (50 mM CH COONa, 50 mM NaCl, 1 mM EDTA, 2 mM DTT), MES
3
at pH 6 (50 mM MES, 30 mM NaCl, 2 mM DTT) and phosphate buffered saline, pH 7.4
supplemented with 2 mM DTT. The substrates were incubated in 96-well white microtiter
plates in two series in a volume of 100 µl and final concentration of 50 µM (for each buffer
system). Cat C was applied at 220 pM. The change in fluorescence intensity was observed
over time (λ =320 nm and λ =400 nm) and recorded on Spectra Max Gemini EM
ex
em
(Molecular Devices, France). The initial hydrolysis rates were measured in triplicate for each
substrate and the results are expressed as the mean ±SD.

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Selectivity assay
For the selectivity assay, the selected substrates at the concentration of 7.5 µM, were
incubated with the different representative cathepsins (Cat C, L, B, X, S, K, A, and V) at
concentration of 600 pM. All enzymes were purchased from R & D Systems (USA). The
proteolytic activity was measured in 25 mM MES buffer containing 50 mM NaCl and 5 mM
DTT, pH 6.0 at 37 °C. The selected compounds were incubated with neutrophil serine
proteases (NSPs): Cat G, HNE and PR3 either under assay conditions described above as well
as using the optimal for NSPs buffer system (100 mM TrisHCl, 500 mM NaCl, pH 7.5).
Substrate scissile bond confirmation
The products of enzymatic reactions were analyzed using two different methods. Firstly,
reaction mixtures were studied by RP-HPLC with fluorescence detection (λ =320 nm and
ex
λem=400 nm; Jasco LC system, Japan) equipped with Gemini 5µm C18 110Å (150×4.6 mm)
column. After 90 minute incubation under conditions described above of the enzyme with
examined substrate, 5 µl of the reaction mixture were injected into the system and a linear
gradient from 10 to 50 % B (where A: 0.1% TFA in H O; B: 80% acetonitrile in A) in 20 min
2
with a flow rate of 1 ml/min was applied. The retention time of the products was compared to
the retention time of the synthesized reference compound Thi-Ala(Mca). In the second
method, selected reaction mixtures were analyzed by LC-MS using AB SCIEX TripleTOF
5
600+ with microLC system (USA) equipped with Eksigent ChromXP C Cl column (120Å,
18
3
µm, 150×0.3mm). The following elution program was used: 0-2 min - 10%B, 2-23 min - 10
to 90% B, 23-28 min - 90% B (where A: 0.1% formic acid in H O and B: 0.1% formic acid in
2
acetonitrile).
Molecular Docking
Protein-ligand docking calculations were carried out using Autodock Vina 1.1.2 (28). The
crystal structure of human cathepsin C complex with the inhibitor (Gly-Phe-diazomethane;
2
DJF.pdb) was selected as a receptor (29). The water coordinates as well as inhibitor
molecule were firstly removed from the structure. The geometry of the Thi-Ala(Mca)-Ser-
Gln-Tyr(3-NO )-NH substrate was optimized using MM2 force field. Receptor and ligand
2
2
polar hydrogens as well as rotatable bonds of substrate were assigned using AutoDockTools
.5.6 (Scripps Research Institute, USA). Molecular docking was performed using default
1
settings with 3D search space defined by the 40×30×26 Å box centered at 25.5, 25.9, 21.8 to
include cathepsin C substrate-binding site.

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Biological studies
Preparation of the neutrophils lysate
Neutrophils were isolated from fresh whole blood of healthy volunteers as described
previously (30). Purified neutrophils were lysed in 50 mM CH COONa, 30 nM NaCl, 2 mM
3
DTT, 1 mM EDTA, 0.5% NP-40, pH 5, at the concentration of 50000 cells in 1 µl. Soluble
fractions were separated from cell debris by centrifugation at 10,000×g for 10 min and the
obtained supernatant was used in the study.
Preparation of urine samples from healthy donors
Healthy urine samples were centrifuged at 3000 g for 15 min at 4°C to eliminate cells and
debris. The urine supernatants were then concentrated 20 times using Vivaspin 15R
concentrators (molecular cutoff, 10 kDa; Sartorius, Goettingen, Germany) and stored at 4°C.
Preparation of the bronchoalveolar lavage fluids
The bronchoalveolar lavage fluids (BALFs) were collected during the course of the ongoing
treatment of patients hospitalized at the Department of Pneumology at the CHRU of Tours,
France, following their consent in accordance with the Helsinki Declaration (2000) of the
World Medical Association. BALFs were collected during bronchoscopy by the injection of
saline in a segmental bronchus in accordance with international recommendations. From the
recovered BALFs the soluble fractions was separated from the cellular fractions by
centrifugation at 1000×g for 10 min followed by second centrifugation step at 3500×g for 15
min in order to remove the remaining membrane fraction from the supernatant. Aliquots of
1
.0 mL of BALFs taken from patients with histiocytosis X (HX) and desqumative interstitial
pneumopathy (DIP) were concentrated 20 times by ultrafiltration using Vivaspin
concentrators with the filtration threshold of 150 kDa (GE Healthcare Bio-Sciences AB,
USA).
Measurements of Cat C activity in biological samples
In the activity assay, the substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH (25 µM) was
2
2
incubated at 37 °C with the sample diluted in 50 mM CH COONa, 30 nM NaCl, 2 mM DTT,
3
1
mM EDTA, pH 5. As control, the sample was preincubated for 30 minutes with 5 µM of
Thi-Phe-CN, the Cat C inhibitor, at 37 °C in the buffer mentioned above. The following
amounts were used: 5 µl of neutrophil lysate, 5 µl of concentrated urine, 3 µl of the
concentrated BALFs . The change of fluorescence intensity was observed over time (λ =320
ex

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nm and λ =400 nm). All measurements were performed in duplicate and recorded on the
em
SpectraMax Gemini EM (Molecular Devices, France).
RESULTS
FRET Pair Selection
In terms of specificity and sensitivity, FRET substrates have proven to be superior over
chromogenic and fluorogenic substrates. The structures of compounds employed here to
develop FRET substrates for the measurement of Cat C activity are presented in Fig. 1.
Firstly, we have approached two different fluorescence donors that could possibly enable us
to measure Cat C activity: o-aminobenzoic acid (ABZ) and β-(7-methoxy-coumarin-4-yl)-L-
alanine (Ala(Mca)). Several findings have already showed the successful application of FRET
pair ABZ/Tyr(3-NO ) for sensitive measurement of different endopeptidases activity (31) (32)
2
(33). Despite the fact that Cat C requires the presence of free N-terminal amino group of the
substrate to be bound with high affinity, Cat C is able to recognize substrates containing the
N-terminal protection such as Z-Phe-Arg-AMC (34) and Sar-Phe-AMC (where Sar is N-
methyl glycine) (6). Considering the preference of Cat C towards large, hydrophobic amino
acid residues in P position, two substrates: ABZ-Phe-Tyr(3-NO )-NH (Fig. 1A) and ABZ-
1
2
2
Bip-Tyr(3-NO )-NH (where Bip is biphenylalanine) were synthesized and studied. Both
2
2
substrates were not hydrolyzed by Cat C; however, they exhibited high affinity towards Cat L
and were further used as lead structures to develop ultrasensitive internally quenched
substrates of this protease (32).
In the light of the obtained results, the fluorescent analogue of alanine, Ala(Mca), was found
more promising as it contains free α-amino group. Three compounds were subsequently
synthesized to assess the utility of this fluorescence donor: Ala(Mca)-Phe-Tyr(3-NO )-NH2
2
(Fig. 1B) Ala(Mca)-Tyr-Tyr(3-NO )-NH and Ala(Mca)-Nle(6-OBzl)-Tyr(3-NO )-NH2
, 2 2 2
(where Nle(6-OBzl) is 6-benzyloxynorleucine). Previously reported substrates with Phe and
Tyr were shown to exhibit high affinity towards Cat C (6) (7), whereas the introduction of
Nle(6-OBzl) in P position was based on previously reported specific Cat C substrate Abu-
1
Nle(6-OBzl)-ACC (7). Unfortunately, none of these compounds was hydrolysed in the
presence of Cat C, wheareas the compound containing Nle(6-OBzl) analogue was the only
one being hydrolyzed after incubation with Cat L.
Due to the large and hydrophobic character of Ala(Mca), this amino acid analogue was next
evaluated in P position. The L-β-2-thienylalanine (Thi) was introduced as the P residue of
2
1

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the substrates in order to increase the specificity and selectivity towards Cat C, as it was
previously shown for potent and selective inhibitors of Cat C (e.g.
L-β-2-thienylalanyl-L-
phenylalanine nitrile (ThiPhe-CN) displaying the IC value of 0.9 ± 0.3 nM (35)). The
5
0
fluorogenic dipeptide substrates bearing P Thi exhibit higher affinity towards Cat C than
2
corresponding analogues with Gly present in same position for routinely used substrates (6)
(
36) (37) (see Fig. 1S). The obtained compound Thi-Ala(Mca)-Tyr(3-NO )-NH (Fig. 1C) was
2 2
efficiently hydrolyzed in the presence of Cat C and not Cat L (the observed hydrolysis rate
was 10 times lower for Cat L than for Cat C) and was further used as a lead structure for the
design of FRET substrate series with extended peptide chains to optimize the interaction with
Cat C primed pockets.
Enzymatic Studies of FRET Peptides with Cat C and Cat L
After selection of Ala(Mca)/Tyr(3-NO ) pair, we have synthesized a series of compounds
2
with a general formula Thi-Ala(Mca)-X ′-Tyr(3-NO )-NH , where X ′ was a set of
1
2
2
1
proteinogenic amino acids except for Cys. In order to assess the affinity of new compounds,
we have run the activity assay with both Cat C and Cat L. Cat L is an ubiquitous protease
which participates not only in the protein breakdown in lysosomes, but is also involved in the
proteolytic activation of various zymogens and prohormones (4) (38). Since in our studies Cat
L was used to activate Cat C and was present at low concentration in the assay solutions, an
incubation of all studied compounds was performed separately in parallel with Cat L as the
control. The results highlighting the differences in the hydrolysis rates of studied substrates in
the presence of both enzymes are presented in Fig. 2A. The enzymatic activity was expressed
as the average relative activity, meaning that Cat C activity observed towards each compound
is presented as a percentage of the hydrolysis rate observed for the best substrate Thi-
Ala(Mca)-Ala-Tyr(3-NO )-NH . The substrate Thi-Ala(Mca)-Ser-Tyr(3-NO )-NH exhibited
2
2
2
2
one of the highest hydrolysis rates with Cat C and at the same time it was not cleaved by Cat
L. To increase the specificity and selectivity of the developed substrates, Ser was selected for
the next series of compounds with general formula Thi-Ala(Mca)-Ser-X ′-Tyr(3-NO )-NH
2,
2
2
where 19 proteinogenic amino acids (except for Cys) introduced as X ′ were evaluated. The
2
results of the enzymatic assay with Cat C and Cat L at pH 6 are presented in Fig. 2B. Among
substrates of this group the highest susceptibility to proteolytic cleavage by Cat C was
observed for compounds containing Gly or Gln in P ′ position which simultaneously were not
2
hydrolyzed by Cat L under the conditions of our assay. Both substrates were further evaluated
including determination of kinetic parameters at pH 5 and 6. Due to the fact that almost all of

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already reported synthetic substrates of Cat C are fluorogenic dipeptides, we focused our
attention on the impact of the chain extension on the kinetic parameters of newly developed
substrates. Unfortunately, due to the low solubility of tripeptidyl substrate Thi-Ala(Mca)-
Tyr(3-NO )-NH its kinetic parameters could not be determined. Instead, for further studies
2
2
we have chosen the fluorogenic dipeptide bearing the same amino acid residue in P position,
2
Thi-Phe-AMC, and the most promising tetrapeptide substrate Thi-Ala(Mca)-Ser-Tyr(3-NO )-
2
NH . The obtained results are summarized in Table 1. All of the substrates displayed the
2
Michaelis constant values in the range of 3.03-5.23 µM at pH 5 and 2.22-3.64 µM at pH 6
revealing similar affinity towards Cat C. Extending the peptide chain of tetrapeptide substrate
with Gly in P ′ resulted in a slight improvement of its affinity, while introducing Gln had no
2
impact on the Michaelis constant value.
The majority of cathepsins belonging to the papain family of cysteine proteases (B, H, L, C,
X, F, O and V) is ubiquitously expressed in human tissues. They share a similar specificity
pattern of their substrate binding pockets and highly conserved active site thus making a
selective detection of any individual cathepsin very challenging. Considering the selectivity as
a crucial factor for newly developed cathepsin substrates, Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-
2
NH and Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO )-NH were incubated separately with different
2
2
2
cathepsins (Cat C, L, B, S, K, V, X and A) at pH 6 (Fig. 3A). The hydrolysis of Thi-
Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH was observed only in the presence of Cat C and Cat B,
2
2
but the proteolysis rate was approximately 5 times lower for the second enzyme. The second
substrate Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO )-NH was found to be less selective since it was
2
2
hydrolyzed by Cat L. The substrate hydrolysis pattern was followed by HPLC analysis with a
fluorescence detection (Fig. 3B). Interestingly, the fluorescent product Thi-Ala(Mca) (t = 12
R
min) was formed only in the presence of Cat C, whereas the incubation of Thi-Ala(Mca)-Ser-
Gly-Tyr(3-NO )-NH with Cat B resulted in the formation of different hydrolysis product (t
R
2
2
=
10 minutes) which corresponds to the Thi-Ala(Mca)-Ser-Gly (as proven by MS analysis,
see Fig. S3).
The kinetic parameters determined using Cat B (Table 2) showed that both substrates display
similar affinity with the KM values one order of magnitude higher than for Cat C. As
expected, they also proved to be less specific towards Cat B than Cat C with 15-20 times
lower specificity constant values.
We have also investigated whether the substrates were susceptible to proteolysis by neutrophil
serine proteases: cathepsin G, elastase and proteinase 3 since they are stored together with Cat
C in primary granules of neutrophils (10), and no proteolysis was observed.

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Molecular Docking
In order to analyze the binding mode of the Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO )-NH substrate
2
2
the molecular docking was performed using human Cat C crystal structure as a receptor (29).
The overall arrangement of P -P ′ residues in the enzyme binding site corresponds to the
2
2
substrate-binding sites organization model proposed for the papain-like proteases (Fig. 4A)
39) (1) allowing for correct positioning of the scissile bond carbonyl carbon close to the
(
catalytic Cys234 residue (Fig. 4B). In presented orientation the side chain of Thi occupies the
S2 binding pocket while its amino-terminal group is located in close proximity to the carboxyl
oxygen atoms of Asp1 side chain and carbonyl oxygen of Gly277 allowing the hydrogen bond
formation (Table 1S). The interactions between the amino-terminal group of the substrate and
the Asp1 and Gly277 of Cat C are responsible for the substrate N-terminus docking and are
crucial for proteolytic activity. The binding of the substrate within the S -S pockets is
1
2
stabilized by hydrogen bonds formed between Thi1 carbonyl oxygen and Gly277 NH group,
and Ala(Mca) NH group with Asn380 backbone CO group (Table 1S).
S′ Subsite Specificity
Two series of FRET peptides were synthesized to study the S′ pockets specificity of Cat C in
detail. Although physiologically Cat C is present inside acidic lysosomes, cytotoxic T
lymphocyte granules (40) and azurophilic granules of neutrophils (41), it is also secreted into
the extracellular environment exhibiting the proteolytic activity at neutral pH values (42) (43).
In this regard, we have examined Cat C prime sites specificity under reducing conditions at
different pH values: pH 5 (acetate buffer), pH 6 (MES buffer) and pH 7.4 (PBS). The
obtained results are presented in Fig. 5. Interestingly, the broadest substrate specificity of Cat
C was observed under acidic environment which mimics the endogenous conditions of Cat C-
mediated protein degradation and zymogen activation. The differences in the affinity
observed for the obtained substrates towards Cat C are more explicit at pH 6 and 7.4. As
shown in Fig. 5A, the highest hydrolysis rates were exhibited by the substrates with polar
amino acid residues Ser, Thr, basic Lys, Arg or Ala and Gly as P ′ residues. Although
1
substrates with aromatic P ′ residues were efficiently hydrolyzed at pH 5 (approximately 50%
1
of the hydrolysis rate observed for substrates with polar amino acid residues), the turnover
number of these substrates with Cat C significantly decreased at pH 6 and 7.4 with the
exception of His at this position. In contrast, the acidic amino acid residues as well as

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aliphatic Val and Ile were not well tolerated in this position. The series of FRET peptides
differing by the amino acid residue present in P ′ position showed the broad specificity of Cat
2
C at pH 5 since we did not see significant differences in their hydrolysis rates. At increased
pH we observed the hydrolysis rate pattern similar to P ′ position. The substrates with Gly and
1
Ala exhibited the highest hydrolysis rate followed by substrates bearing polar and basic amino
acid residues although the differences were less noticeable than for P ′ position. The
1
comparison of the best substrate hydrolysis rates of each series at different pH values can be
found in the Supplementary Information associated with this manuscript (Fig. 2S).
Biological Studies
Cat C is present at high level in spleen, liver, kidneys and lungs (44) as well as in
hematopoietic cells (41), and can be secreted into the extracellular environment by mast cells,
cytotoxic T lymphocytes and neutrophils (42) (43) (24). Considering the high abundance of
mature Cat C in human tissues and body fluids, we have applied newly developed selective
substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH for the measurement of Cat C activity in
2
2
biological samples of different origin. An efficient substrate proteolysis was observed when
incubated in buffered solution of human neutrophil lysate obtained from 250 000 cells (Fig.
6
A) while lysis of 5000 cells was found to be sufficient for the detection of Cat C activity. In
order to confirm that the observed signal results from the activity of Cat C, the lysate was
preincubated with potent Cat C inhibitor Thi-Phe-CN (35) prior the activity assessment. The
results showed that only 3% of an initial Cat C activity was observed, suggesting high
selectively of developed substrate and its proteolytic resistance to the action of irrelevant
proteases present in neutrophil cell lysate. Subsequent RP-HPLC analysis with fluorescence
detection (Fig. 6B) of both reaction mixtures along with MS analysis (m/z 417.1123; Fig. 4S)
confirmed the formation of Thi-Ala(Mca).
The Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH was also applied for the detection of Cat C
2
2
activity in lysates of three human cell lines where Cat C activity has already been reported
such as PLB-985 (human myeloid leukemia cell line), U937 (human leukemic monocyte
lymphoma cell line) and THP-1 (human acute monocytic leukemia cell line) (24). Similarly to
our findings on human neutrophil cell lysates, the activity of Cat C in cell line lysates was
inhibited by Thi-Phe-CN and confirmed by the formation of same dipeptide fluorescent
product (Fig. 5S and 6S).
We have further examined if the Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH substrate could be
2
2
utilized for the selective measurement of Cat C activity in pulmonary secretions. Until now it

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has been only confirmed that proteolytically active Cat C is present in the bronchoalveolar
lavage fluids (BALFs) from patients with neutrophilic lung inflammation, but not in BALFs
from healthy individuals (24). Although Cat C is stored intracellularly by alveolar
macrophages in high amounts, only little or none could be detected in BALFs from patients
with macrophage-dominated lung inflammation as analyzed by Western blot or Gly-Phe-
AMC-based activity assay. Herein, we have investigated whether Thi-Ala(Mca)-Ser-Gly-
Tyr(3-NO )-NH could be useful for the detection of mature Cat C in BALFs collected from
2
2
patients with histiocytosis X (HX) and desquamative interstitial pneumopathy (DIP), chronic
inflammatory respiratory diseases characterized by the predominance of macrophages. As
shown in Figure 7, the substrate was efficiently hydrolyzed in buffered solutions of BALFs
from patients with HX (n = 6) and DIP (n = 3) and the observed proteolytic activity was
significantly reduced after preincubation of BALFs with Cat C-specific inhibitor Thi-Phe-CN.
Similar to above, all reaction mixtures were analyzed by RP-HPLC confirming the formation
of expected hydrolysis product Thi-Ala(Mca), whereas significantly lower signal was
observed when Cat C was inhibited by Thi-Phe-CN (Fig. 7S and 8S). Additionally, using the
calibration curve obtained by the titration of the substrate with recombinant Cat C, we have
estimated the level of Cat C in BALFs to be in picomolar range (Table 3).
It has been recently demonstrated that Cat C is secreted by bladder epithelial cells of healthy
humans. As presented in Fig. 8, the activity of CatC in buffered human urine can be
specifically selectively measured with the developed substrate.
DISCUSSION
Herein, we have presented the design, synthesis and enzymatic characterization of the first
FRET substrates of human Cat C. During the course of the study, we have synthesized and
evaluated 44 novel compounds of which the most potent substrate Thi-Ala(Mca)-Ser-Gly-
Tyr(3-NO )-NH was successfully applied for the specific detection of Cat C activity in
2
2
various biological samples.
Due to the aminodipeptidase activity of Cat C almost all of already reported synthetic
substrates are fluorogenic dipeptides, comprising both, natural and non-proteingenic amino
acids (6) (7). The binding preference of Cat C has also been investigated by several research
groups focused on potent Cat C inhibitor development (45) (35) (46). As a result, the non-
prime site (S and S pockets) specificity of human Cat C has been extensively studied using
2
1

ACCEPTED MANUSCRIPT
compounds with structurally diverse amino acid residues. Although a very broad specificity of
human Cat C was originally postulated, it was found to be more restricted than previously
claimed (6) (7). It has been found that human Cat C prefers small and aliphatic amino acid
side chains such as Ala, Abu and Met in P and bulky, aromatic residues well accepted in P
2
,
1
position. One report on Cat C-catalyzed hydrolysis of dipeptide substrates employing the
steady-state and pre-steady state kinetic methods showed that the amino acid residue present
in the P position is the main determinant of catalysis (47). While most of the investigated Cat
1
C inhibitors are mimics of dipeptide substrates bearing thiol-reactive groups, inhibitors
addressing the S′ binding pockets in addition to the S subsites also have been reported. Horn
et al. used peptide combinatorial chemistry approach to study the binding preference of the
sites S -S ′, and find amino acid residues enabling effective Cat C inhibition (48). It was
2
1
established that oligoarginines exhibit high inhibitory potency, that improves with the
elongation of peptide chain reaching the maximum for octa- -arginine, for which the binding
mode spanning from S to S ′ subsites was suggested. Bondebjerg et al. developed Cat C
L
2
4
inhibitors based on a semicarbazide scaffold occupying S -S ′ subsites with various aromatic
2
2
groups introduced in P ′ and P ′ positions (49). In 2013 (2S,3S)-oxirane-2,3-dicarboxylic acid
1
2
hydrazide-based Cat C inhibitors were reported by Radzey et al. (50). In the designed series
of the inhibitors S ′ and S ′ pockets were occupied by leucine residue and iso-amyl moiety,
1
2
respectively. In our study we have designed and synthesized the first FRET substrates of Cat
C, that allowed for detailed investigation of the S′ subsite specificity of Cat C. Initially Cat C
was considered to have very broad specificity thus a technique applying Cat C for amino acid
sequence determination was developed (51). However, the structure of the N-termini of Cat C
physiological substrates is very restricted and similar for all recognized molecular targets with
Ile in P ′ and Ile or Val in P ′ position (Table 2S). We expected to see similar binding
1
2
preference for substrates bearing these amino acid residues in corresponding positions. To our
surprise, we have found that substrates with polar and positively charged side chain amino
acid residues are preferred in P ′ position and, although the tetrapeptide substrates were less
1
efficiently cleaved at higher pH, the preference for such amino acid residues was still the most
pronounced at pH 7.4. While the compound Thi-Ala(Mca)-Ile-Tyr(3-NO )-NH was
2
2
hydrolyzed at pH 5, it was not cleaved at pH 7.4 at the same enzyme concentration. The
results of Cat C S ′ specificity showed that all pentapeptide substrates were efficiently cleaved
2
at pH 5 suggesting that with an optimal P -P ′ sequence, the residue occupying P ′ position
2
1
2
does not influence the overall substrate specificity towards Cat C. Nevertheless, we have
shown that the extension of the peptide chain with Gly or Gln in P ′ position enhanced the
2

ACCEPTED MANUSCRIPT
substrate turnover as compared to Thi-Ala(Mca)-Ser-Tyr(3-NO )-NH resulting in increased
2
2
sensitivity of Cat C activity measurement.
Molecular docking of the Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO )-NH was performed in order to
2
2
study the binding mode of the developed pentapeptide substrates in the active site of Cat C.
The hydrogen bond network observed in the presented model corresponds to the interactions
found in cathepsin C complexed with its inhibitor (29). In contrast to the deep, well defined S2
binding pocket Cat C does not possess distinct, well-structured S ′ and S ′ substrate-binding
1
2
sites. The S binding cavity is rather shallow and located above the central helix and catalytic
1
Cys residue. The S ′ substrate-binding groove is created by the loop spanning Gln228-Ser233
2
residues (3). Such open character could partially explain the observed broad specificity
toward the P ′ substrate residues, mainly amino acids containing small or hydrophilic side
2
chains. In addition, the low hydrolysis rate of substrates containing acidic side chains at P2′
position may by the result of their close proximity to the carbonyl oxygen atoms of the S2′
loop backbone. Although the S ′ substrate-binding pocket located above the catalytic His
1
residue is mainly hydrophobic, nevertheless, the imidazole ring may promote the acceptance
of hydrophilic residues such as Ser or Thr at P ′ position.
1
The comparison of Cat C structure to the structures of other cathepsins showed no unique
features of Cat C on the substrate binding sites surface (S , S ′ and S ′) (3). However, the
1
1
2
previous S′ subsite specificity analyses of Cat L and Cat B indicate the presence of several
differences that could potentially be used for the design of selective Cat C substrates (32) (52)
(53), not recognized by Cat L and Cat B, proteases widely expressed by various human
tissues. Substrates containing Arg and Lys are well accepted in P ′ position by Cat C, but not
1
Cat L, which prefers Ser, Thr and acidic amino acid residues, as well as their amides (32). In
case of the pentapeptide substrates of general formula Thi-Ala(Mca)-Ser-X ′-Tyr(3-NO )-
2
2
NH presented in this study, we have found that some of them were cleaved by Cat L after
2
carboxylic side of Ser thus, in order to find more selective Cat C substrates, we have further
focused on the differences between binding preference of S ′ subsite of Cat C and S ′ subsite
2
1
of Cat L. The resulting substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH was not cleaved by
2
2
Cat L under conditions of our assay, which is in agreement with our previous results where
the Cat L substrate with Gly in P ′ was not efficiently hydrolyzed in the presence of the
1
enzyme (32). The primary goal of current study was to obtain Cat C substrates displaying an
increased selectivity over Cat L. Since both proteases coexist in biological samples (urine,
BALFs) studied in this work, we have selected Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH to be
2
2

ACCEPTED MANUSCRIPT
used in biological studies. On the other hand, the developed substrate was hydrolyzed by Cat
B after carboxylic side of Gly, which is in good agreement with the results of proteomic
identification of protease cleavage site carried out for Cat B specificity characterization which
showed Gly to be one of the preferred amino acid residues in P (52).
1
Several mechanisms have developed in order to prevent organism from harmful, uncontrolled
proteolysis including enzyme compartmentalization inside the lysosomes, the synthesis of
proteases as zymogens, pH control, regulation of enzyme activity by various endogenous
inhibitors or combination of all these factors (1). When Cat C is secreted outside the cell, its
activity is significantly decreased as compared to the acidic pH, nevertheless, under
pathological conditions of tumor environment or during inflammation its expression is often
increased, so for a relatively short time the significant proteolysis may occur. Moreover, the
acidic pH is often found at the sites of inflammation when the neutrophils are present (54).
Considering all of above, we have examined the developed Cat C substrates at different pH
values. Our findings suggest that at increased pH some of Cat C targets may be protected
from proteolysis due to the decreased Cat C substrate binding as well as due to observed pH-
dependent S′ specificity, which may participate in the defense mechanism controlling an
undesired proteolytic activity. Nevertheless, the significance of this finding needs to be
further evaluated in detail. The pH-dependent substrate specificity has been previously
reported for Cat B, but it resulted from different mechanism of protease action switching from
carboxydipeptidase at pH 4.5-5.5 to endopeptidase activity at approximately pH 7.4 (53). It
has been previously reported that at pH close to 7.5 the major activity of Cat C is
transamidation leading to the synthesis of long-chain peptides from dipeptide amides and
esters (55) (56). However, in case of our pentapeptide substrates, we have observed only the
formation of Thi-Ala(Mca).
SUMMARY
The first FRET substrates of Cat C, allowing the detailed S′ subsite specificity study of the
enzyme, were designed and synthesized. The substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-
2
NH was applied for detection of Cat C activity in various cell lysates, urine and BALF
2
samples.
AUTHOR CONTRIBUTION
Monika Łęgowska, Adam Lesner and Brice Korkmaz participated in the research design.
Monika Łęgowska, Anna Wojtysiak, Yveline Hamon and Renata Grzywa conducted the

ACCEPTED MANUSCRIPT
experiments. Timo Burster contributed materials and provided expertise in enzymology.
Monika Łęgowska, Adam Lesner, Krzysztof Rolka, Brice Korkmaz, Renata Grzywa and
Marcin Sieńczyk performed data analysis. Monika Łęgowska wrote the manuscript, while
Adam Lesner, Brice Korkmaz and Marcin Sieńczyk contributed to the writing process.
FUNDING
The presented study was supported by the National Science Centre (NCN) in Poland (UMO-
2
012/07/N/ST5/00128).
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Table 1. Kinetic characterization of the obtained Cat C substrates.
pH 5
pH 6
-5
-5
Cat C
-1
k /K × 10
-1
k /K × 10
cat M
cat
M
K [µM]
M
k [s ]
-1 -1
K [µM]
M
k [s ]
-1 -1
cat
cat
[
M ×s ]
[M ×s ]
Thi-Phe-
AMC
3
.03±0.01 2.07±0.05
6.84±0.18
3.64±0.27 3.5±0.01
9.64±0.73
a
P1′=Ser
P2′=Gly
5.16±0.56 2.51±0.08
3.21±0.24 4.29±0.1
5.23±0.23 6.19±0.59
4.91±0.70
13.4±0.7
11.8±1.0
3.26±0.31 1.83±0.23
2.22±0.12 3.45±0.08
3.36±0.29 5.45±0.35
5.60±0.38
15.6±0.5
16.6±0.6
b
c
P2′=Gln
a
b
c
Thi-Ala(Mca)-Ser-Tyr(3-NO
Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO
Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO
2
)-NH
2
2
)-NH
2
2
)-NH
2

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Table 2. Kinetic characterization of the obtained substrates with Cat B.
pH 5
pH 6
-5
-5
Cat B
-1
k /K × 10
-1
k /K × 10
cat M
cat
M
K [µM] k [s ]
-1 -1
K [µM] k [s ]
-1 -1
M
cat
M
cat
[
M ×s ]
[M ×s ]
0.84±0.04
0.70±0.02
a
P2′=Gly 20.4±0.5 1.94±0.02
P2′=Gln 22.6±1.0 1.76±0.17
0.95±0.02
0.78±0.04
17.2±0.5 1.44±0.11
20.1±0.1 1.40±0.06
b
a
Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO
Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO
2
)-NH
)-NH
2
b
2
2
Table 3. Concentration of the tetrameric Cat C in the BALFs from patients with histiocytosis
X (HX) and desquamative interstitial pneumopathy (DIP).
Cat C tetr
Sample
[pM]
HX 1
HX 2
HX 3
HX 4
HX 5
HX 6
DIP 1
DIP 2
DIP 3
16.8
18.0
3.6
70.7
7.0
30.5
9.5
17.5
22.6
Fig. 1. Structures of compounds tested for the selection of an optimal fluorescence resonance
energy transfer donor/acceptor pair. The expected cleavage site is indicated by an arrow.

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Fig. 2. Enzymatic activity of Cat C and Cat L after incubation with the substrates: A) Thi-
Ala(Mca)-X ′-Tyr(3-NO )-NH with 0.85 nM Cat C and 0.21 nM Cat L, and B) Thi-
1
2
2
Ala(Mca)-Ser-X ′-Tyr(3-NO )-NH with 0.21 nM Cat C and 0.04 nM Cat L; the data is
2
2
2
presented as the average relative activity. The hydrolysis rate was normalized to the best
substrate in each series (X ′= Ala with Cat C and X ′=Arg with Cat C) and are given as
1
2
percentages ± SD.

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Fig. 3. Selectivity of Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH action. A) Comparison of
2
2
proteolysis velocity observed in the presence of Cat C, Cat B and Cat L. B) RP-HPLC
analysis with fluorescence detection of the reaction mixture after 2 hours incubation of the
substrate with different cathepsins: 1 – Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH ; 2 – Thi-
2
2
Ala(Mca); 3 – Thi-Ala(Mca)-Ser-Gly; 4 – Thi-Ala(Mca)-Ser.
Fig. 4. The model of Thi-Ala(Mca)-Ser-Gln-Tyr(3-NO )-NH substrate binding by human
2
2
cathepsin C (based on 2DJF.pdb). A) The substrate-binding site is presented as a surface with
S pocket in red, S – blue, S ′- green, S ′ – yellow (1). B) Visualization of highly conserved
2
1
1
2
residues of the cathepsin C active center (29) and hydrogen bonds formed with Thi-Ala(Mca)-
Ser-Gln-Tyr(3-NO )-NH substrate.
2
2

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Fig. 5. Profiling of Cat C specificity in positions P ′ (A) and P ′ (B) at different pH values.
1
2
The Cat C activity is expressed as the average relative activity ± SD, the percentage of the
highest hydrolysis rate observed in each assay.

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Fig. 6. A) The Cat C activity assayed with Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH in the
2
2
human neutrophil lysate preincubated with (+) or without (-) Cat C inhibitor (Thi-Phe-CN, 5
µM). B) RP-HPLC analysis of the Cat C activity in the human neutrophil lysate reaction
mixtures of A).
Fig. 7. Cat C activity detected with Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO )-NH in the BALF
2
2
samples collected from the patients suffering from the chronic inflammatory respiratory
diseases characterized by the predominance of macrophages: histiocytosis X (HX) and
desquamative interstitial pneumopathy (DIP). Both non-treated (-) and preincubated with 5
µM Thi-Phe-CN samples were used (+).