Three Wavelength Substrate System of Neutrophil Serine Proteinases

Article pubs.acs.org/ac

Three Wavelength Substrate System of Neutrophil Serine Proteinases Magdalena Wysocka,† Adam Lesner,*,† Natalia Gruba,† Brice Korkmaz,§ Francis Gauthier,§ Mizuki Kitamatsu,‡ Anna Łęgowska,† and Krzysztof Rolka† †

Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Department of Medical and Bioengineering Science, Okiyama University, 3-1-1 Tsushimanaka kita-ku Okayama, Okayama, Japan § INSERM U1100, Faculté de Médecine de Tours, Université François Rabelais in Tours, France ‡

S Supporting Information *

ABSTRACT: Neutrophil serine proteases, including elastase, proteinase 3, and cathepsin G, are closely related enzymes stored in similar amounts in azurophil granules and released at the same time from triggered neutrophils at inflammatory sites. We have synthesized new fluorescence resonance energy transfer (FRET) substrates with different fluorescence donor−acceptor pairs that allow all three proteases to be quantified at the same time and in the same reaction mixture. This was made possible because the fluorescence emission spectra of the fluorescence donors do not overlap and because the values of the specificity constants were in the same range. Thus, similar activities of proteases can be measured with the same sensitivity. In addition, these substrates contain an N-terminal 2-(2-(2-aminoethoxy)ethoxy)acetic acid (PEG) moiety that makes them cell permeable. Using the mixture of these selected substrates, we were able to detect the neutrophil serine protease (NSP) activity on the activated neutrophil membrane and in the neutrophil lysate in a single measurement. Also, using the substrate mixture, we were in a position to efficiently determine NSP activity in human serum of healthy individuals and patients with diagnosed Wegener disease or microscopic polyangiitis.

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(mainly, Wegener granulomatosis), and Papillon-Lefevre syndrome.5 Amino acid residues in the substrate, which interact with the protease pockets, are referred to (based on the Schechter Berger notation6) as P1, P2, P3, ..., Pn and P1′, P2′, P3′, ..., Pn′. Corresponding enzyme substrate binding pockets, termed nonprime (S1, S2, S3, ..., Sn) and prime (S1′, S2′, S3′, ..., Sn′) sites depending on their proximity to the active center, are present.6 One method for detection of protease activity is to introduce chemical moieties into the peptide chain so that one serves as a donor of fluorescence and the second acts as a quencher. The presence of both in a single peptide chain results in fluorescence resonance energy transfer, called FRET.7 Upon enzymatic hydrolysis of peptide bonds (when the enzyme displays affinity toward an amino acid sequence), the distance between donor and quencher increases and a fluorescence boost is observed. This phenomenon is commonly utilized by several research groups for determination of interactions between protease and its substrates or protein−protein interactions.

eutrophils express a cell-type specific set of neutrophil serine proteases (NSPs), namely, cathepsin G (CG), proteinase 3 (PR3), and human neutrophil elastase (HNE), which are stored in the cytoplasmic azurophilic granules. Upon neutrophil activation, they are released from the cell. PR3 and HNE are two closely related enzymes with overlapping substrate specificities, which are different from that of CG. All three NSPs are involved in antimicrobial defense by degrading pathogens in the special mechanism called the neutrophil extracellular trap (NET). Among many other functions assigned to these enzymes, PR3 and HNE were also suggested as playing a crucial function in granulocyte development in the bone marrow, apoptosis, blood pressure regulation (CG), antigen processing (CG), platelet activation, degradation of extracellular matrix proteins (HNE, PR3), activation of protease receptor 2 PAR-2, and so on.1−3 NSP activity in healthy humans is regulated and controlled by a set of cognate inhibitors, for example, the secretory leukocyte protease inhibitor, serpin family (α1 protease inhibitor, α1 antichymotrypsin), elafin, and finally and nonspecifically, macroglobulins.4 However, when NSPs escape from tight and strict control, they become invaders and destructors within a human body. Thus, many pathologies are associated with uncontrolled NSP action, e.g., chronic inflammatory lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome), antineutrophil cytoplasmic autoantibody associated vasculitides © 2012 American Chemical Society

Received: June 18, 2012 Accepted: July 16, 2012 Published: July 16, 2012 7241

dx.doi.org/10.1021/ac301684w | Anal. Chem. 2012, 84, 7241−7248

Analytical Chemistry

Article

on an RP-HPLC Pro Star system (Varian, Australia) equipped with a Kromasil 100 C8 column (8 × 250 mm) (Knauer, Germany) and a UV−vis detector. A linear gradient from 10 to 90% B was applied within 40 min (A: 0.1% TFA; B: 80% acetonitrile in A). The analyzed peptides were monitored at 226 nm. Mass spectra of the synthesized peptides were recorded using a Biflex III MALDI TOF mass spectrometer (Bruker Daltonics, Germany) and α-cyano-4-hydroxycinnamic acid as a matrix. Fluorescence Studies. UV and fluorescence spectra of fluorescent derivatives were recorded for kinetic studies in the microplate reader. A FluoroSTAR (BMG, Germany), equipped with a set of 12 extinction emission filters, was used. Enzymatic Studies. The human enzymes cathepsin G, neutrophil elastase, and proteinase 3 (called together neutrophil serine proteinases, NSPs) were used in this study. Proteinase 3, cathepsin G, and human neutrophil elastase were supplied by Elastin Inc. (USA). Bovine β-trypsin and turkey ovomucoid third domain (OMTKY3) were supplied by Sigma Aldrich (Germany). The concentration of bovine β-trypsin stock solution was determined by titration with NPGB (nitrophenol-p-guanidino benzoate) using burst kinetics. The concentration of each of the NSP members was determined using a standardized solution of common inhibitor (OMTKY3), previously titrated by a solution of bovine βtrypsin as was described previously.15,16 Energy Transfer Efficiency Determination. For selected substrates, fluorescence increase and energy transfer efficiency were determined according to eqs I and II, respectively: where F0 is the fluorescence of the intact peptide and F1 is the fluorescence of the product after complete hydrolysis. F0 and F1 were derived after the subtraction of buffer autofluorescence (Fbuff) from both the initial (Fini) and final fluorescence (Fmax).

In the past years, we focused our research on peptides displaying FRET and their utilization as substrates of several important proteases8,9 with emphasis on NSP members.10−12 The main idea of our investigation was, using multiple approaches including combinatorial chemistry13 and inhibitor based design,14 to obtain highly specific FRET peptides that would selectively interact with one of the neutrophil serine proteases. With this aim, the specific substrates were developed for HNE,11 PR3,12 and CG.15 In our previous work, we described a low molecular weight substrate (Ala(Box(Pyr))Tyr-Tyr-Abu-ANB-NH2) of PR3 (where Ala(Box(Pyr)) stands for N-methylpyrrole benzoxazole-L-alanine) which allowed us to detect activity (in a subnanomolar range) of this enzyme in human serum of healthy individuals and Wegener-diagnosed patients.16 Moreover, we found a correlation between the level of anticytoplasmic antibodies (cANCA) that is a key factor for Wegener diagnosis and the activity of PR3. As mentioned above, all previously described methods for NSP activity determination in biological material containing multiple enzymes apply a single substrate that results in detection/ quantification of a particular protease. This is a serious shortcoming in cases when a fast response is required and/or a minimal volume of material is available. The aim of this study was to design, synthesize, and biochemically characterize a substrate system which would enable three neutrophil serine protease activities to be followed using a single measurement. To work properly, such a system, consisting of a mixture of three substrates, should fulfill several requirements: (i) each substrate should display FRET in an intact form, (ii) each substrate should contain one cleavage site, selectively digested by one NSP member only, (iii) upon cleavage by a particular NSP, a substrate should emit fluorescence with λmax distinctly different from the remaining two, which should allow for simultaneous recording of the individual signal, and (iv) if possible, substrates should be cell permeable.

Fmax − Fbuff F = 1 Finit − Fbuff F0

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(I)

⎛ F⎞ energy transfer efficiency (%) = ⎜1 − 0 ⎟ × 100% F1 ⎠ ⎝

EXPERIMENTAL SECTION Chemistry. All peptides were synthesized manually by means of the solid-phase method using Fmoc chemistry, as described previously.12 2-Chloro-chlorotrityl resin (substitution 1.44 meq/g) (RAPP Polymere, Germany) was used as a solid support. The fluorescent amino acids were synthesized according to the procedure described earlier.17,18 In the first step, the Fmoc-protected derivate of the appropriate fluorescent amino acid was dissolved in DCM and incubated for 2 h followed by an MeOH wash. The peptide chain was elongated using the appropriate amino acid derivatives via the N,N'-Diisopropylcarbodiimide/N-Hydroxybenzotriazole (DIPCI/HOBt) method. The introduction of Fmoc-protected PEG amino acid on N-termini of synthesized peptides was achieved using the reaction mixture consisting of Fmoc-PEGOH/HATU/HOAT/DIPEA (1:1:1:2, eqv.), where PEG represents 2-(2-(2-aminoethoxy)ethoxy)acetic acid, HATU represents 2-(7-Aza-1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOAT represents 1-Hydroxy-7azabenzotriazole, and DIPEA represents N,N-Diisopropylethylamine. After completing the synthesis followed by Fmoc removal, the peptides were cleaved from the resin together with the removal of all side chain functions using a mixture of trifluoroacetic acid (TFA)/phenol/triisopropylsilane/H2O (88:5:2:5, v/v). All synthesized peptides contain an amide moiety at their C-termini. Purity of the peptides was checked

(II)

pH Studies. Water based buffers (50 mM) supplemented with 500 mM NaCl were prepared. The pH range was as follows 3, 4, 5, 6, 7, 7.5, 8, 9, and 10 using the same composition for all buffer solutions: 100 mM buffer solution + 500 mM NaCl. For details, see Table 1. Proteolytic Cleavage Pattern Determination. Selected substrates (1.4 μM) were mixed with a 2-fold molar excess of the appropriate experimental enzyme in a buffer used for kinetic studies. HPLC analysis of this mixture was carried out after the following incubation times: 0 and 15 min and 1 h. A Table 1 pH 3 4 5 6 7 7.5 8 9 10 7242

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

M M M M M M M M M

C6H8O7 × H2O/0.2 M Na2HPO4 × H2O CH3COOH/0.1 M CH3COONa CH3COOH/0.1 M CH3COONa MES/1N NaOH MOPS/1N NaOH TRIS-HCl HEPES/1N NaOH TRIS-HCl CHAPS/1N NaOH



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Figure 1. The chemical structures of fluorescent amino acids with their extinction and emission wavelengths.

substrate or substrate mixture was added; the whole plate was mixed thoroughly, and fluorescence increase was followed for 30 min. Ten microliters of PMSF solution was added into the well containing 6 × 105 cells and 100 μL of buffer to achieve a final concentration (100 nM in the well), and the entire plate was incubated at 37 °C for 15 min. Human Serum Studies. Human sera from healthy individuals (27), Wegener disease (12), and microscopic polyangiitis (17) patients were assayed using an equimolar mixture of substrates. A Nalgene 96 (Nalgene, USA) well plate was used. Into each well, 150 μL of serum was mixed with 150 μL of buffer (100 mM Tris-HCl, pH 7.5, supplemented by 500 mM NaCl) followed by adding 1 μL of substrate (each at a concentration of 0.8 μM). The fluorescence emission at three different wavelengths (BAD λem = 490 nm, HOC λem = 395 nm, and HMC λem = 445 nm) was measured for up to 900 s. Each sample was run in triplicate. Flow Cytometry. The 3 × 106 nonactivated neutrophils were resuspended in 3 mL of PBS buffer, and 5 μL of HNE substrate (3.5 μM) was added and mixed thoroughly. At indicated time points (0, 5, and 20 min), 1 × 106 cells were collected and washed 3 times with PBS and finally resuspended in 1 mL of PBS and put in an ice bath. Next, the cells were analyzed on an EPICS XL Flow Cytometer (Beckman Coulter, USA) using the fluorescein isothiocyanate (FITC) filter system.

linear gradient from 5 to 80% B within 30 min was applied (A: 0.1% TFA; B: 80% acetonitrile in A). The analyzed peptides were monitored at 226 nm. The appearing peaks were collected and analyzed using MALDI MS as described above. Sensitivity Curve. A constant amount of a selected peptide (2.8 μM) was added into a buffered solution of a specific enzyme in an appropriate buffer. The amount of the assayed enzyme ranged from 1.1 × 10−8 M to 0.7 × 10−12 M. The fluorescence increase at 395, 445, and 490 nm versus time was measured. All obtained values were measured against a substrate solution with no enzyme added as a control. The threshold limit for all measurements was 3:1 expressed as a signal-to-noise ratio. Kinetic Investigations. For each peptide (1−9), the titration curve for the donor of fluorescence was obtained. Simply, the BAD, HOC, and HMC fluorescence was measured as a function of its concentration in 0.1 M Tris HCl buffer (pH 8.3). These data were used to calculate the concentration of the peptide-containing donor. Then, to the appropriate buffered enzyme solution (at level of 5 × 10−9 M (PR3 and HNE) and 5 × 10−8 M (CG)) increasing concentrations (from 100 nM to 100 μM) of the individual peptide under study were added. The increase of fluorescence for each system was recorded for 10 min. Neutrophil Studies. The neutrophils were purified from fresh blood using Ficol and multiple centrifugation. Neutrophil activation was achieved using a 50 μM solution of CaCl2/ MgCl2 (equimolar) for 2 min. For each microplate experiment, 3.5 to 70 × 104 cells were used. Cells were transferred into the well and then mixed with PBS buffer, and finally, 5 μL of substrate or substrate mixture was added. The whole experiment was performed at 37 °C. The plate was shaken before measurement for 30 s. The increase in the fluorescence of the donor (BAD λem = 490 nm, HOC λem = 395 nm, and HMC λem = 445 nm) was measured for 30 min. Neutrophil Lysates. Human neutrophils were purified from 16 mL samples of peripheral blood collected from healthy volunteers and WG patients into EDTA-containing tubes. The cells (5 to 40 millions) were lysed in 150 or 600 μL of 50 mM HEPES supplemented by 0.75 M NaCl, 005% NP-40, pH = 7.4, and the supernatants were retrieved after centrifugation at 10 000g for 10 min. Inhibitory Study. PR3 selective inhibitor (5 μL; ABZVADaza(nor)VADYQ-Y(NO2)) at a concentration of 20 μM was added to each well containing 6 × 105 cells and 100 μL of buffer. The plate was incubated at 37 °C for 20 min. The

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RESULTS First, we chose the peptide sequences selectively recognized and cleaved by one enzyme only. For further modification, we decided to use the substrate sequences of cathepsin G (MCAVal-Thr-Gnf-Ser-Asp-ANB-NH215), proteinase 3 (ABZ-TyrTyr-Abu-ANB-NH212), and human neutrophil elastase (ABZAla-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln-EDDnp)11 that have been previously developed by our research groups. In the next step, we chose three pairs of suitable, synthetic, fluorogenic amino acids (see Figure 1). Each pair contained a chemical moiety acting as a donor of fluorescence and a second one as an acceptor of fluorescence. Spectral properties of the amino acids used are shown in Figure 2. It worth noticing that the chosen compounds (Lys(Hmc) (2A), Lys(Moc) (2B), Ala(Bad) (2C)), when excited, emitted fluorescence at distinctly different wavelengths (400, 450, and 500 nm, respectively) that was absorbed by quenchers (Orn(Cm3) (2A), Lys(Hoc) (2B), and Lys(FAM) (2C), respectively). After this selection process, nine peptides were designed and synthesized using the solid-phase method applying Fmoc 7243



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Subsequently, all nine compounds were subjected to enzymatic studies. The kinetic parameters (kcat, KM, and kcat/ KM) for each substrate were determined and are listed in Table 2. Substrates with the highest values of the specificity constant (kcat/KM) for a particular enzyme were subjected to further studies. Peptide 1 was selected for PR3, peptide 6 for HNE,and peptide 8 for CG. It is worth mentioning that the obtained substrates have specificity constants ranging from 52.41 × 104 to 25.60 × 103 M−1 × s−1. Next, for equimolar mixtures of substrates 1, 6, and 8, the kinetic parameters were determined using a single protease. We observed no significant changes in the kinetic parameters, neither KM nor kcat (data not shown). An example of a Michaelis−Menten fit for a selected substrate is shown in Figure 6 in the Supporting Information section. For the above selected peptides, the efficiency of energy resonance transfer between donor and acceptor was determined. The energy efficiency was the highest for the HOCMOC pair (51%) (compound 1), and only slightly lower for the two remaining ones: HMC-CM3 (48%, compound 6) and BAD-FAM (40%, compound 9). The same measurements were repeated for equimolar mixtures of three compounds selected earlier: the three peptides each with different combinations of fluorophores were used. As expected, due to unspecific quenching in such a spectrally complicated system, we observed lower (from 6 to 9%) transfer efficiency (see Table 1 in Supporting Information). We would like to stress that such unspecific quenching did not affect the selectivity of the obtained peptides but only increased the background that was observed, thus affecting the limit of detection for a particular protease as compared to the single substrate. Selected substrates that were chosen for pH studies. The specificity parameters (kcat/KM) were determined for each protease substrate system. The experiments were conducted in several buffers where the pH varied from 3 to 10. The obtained specificity pH dependency clearly indicates that all the tested compounds are most efficiently proteolyzed in pH between 7 and 8 (see Figure 3). However, even in pH 5, an environment characteristic for neutrophil lysosome, some residual proteolysis at a level of 10−14% of the maximal is observed for all substrates. Before proceeding with cell studies, the selectivity assay for each substrate (1, 6, and 8) was performed and products of proteolytic cleavage for each peptide were identified (See Supporting Information Figures 1, 2, and 3). As a result of this experiment, we proved that there is no unwanted cleavage of a substrate upon its incubation (up to 1 h) with NSP mixtures that simulate the situation occurring on the neutrophil surface and in human serum. We also performed a titration of all selected substrates with protease to determine the minimal amount (measurable fluorescence output) of protease detected. All substrates were hydrolyzed by the appropriate enzyme at a nano- or subnanomolar range (8 ± 1 nM of PR3, 734 ± 58 pM of HNE, 45 ± 3 nM of CG for substrates 1, 6, and 8, respectively). The titration curves are presented in Figure 4 in the Supporting Information section. Incubation of selected substrates with cognate enzymes results in a huge fluorescence increase (Figure 6A, Supporting Information). Similar results were obtained when equimolar mixtures of three substrates were used and incubated with a single protease (in Figure 6B, Supporting Information, HNE was used). Such results allowed us to proceed to the next step of our studies on human neutrophils. In order to do so, the

Figure 2. Superposition of normalized UV (solid line) and fluorescent spectra (dashed line) of donor moiety and UV spectra of acceptor moiety (dotted line).

chemistry. For each protease, three sequences flanked by three pairs of donor−acceptor residues were obtained. They were analyzed using HPLC and mass spectrometry to confirm their identity (see Table 2). In order to increase the solubility of the synthesized compounds and their ability to cross a cellmembrane, 2-(2-(2-aminoethoxy)ethoxy)acetic acid (PEG) was attached at their N-termini. It is worth mentioning that nonpegylated peptides were hardly dissolved in any water based buffer. 7244



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Table 2. Physicochemical and Enzymatic Characteristics of Synthesized Substrates sequencea (1) (2) (3) (4) (5) (6) (7) (8) (9)

PEG-HOC-Tyr-Tyr-Abu-MOC PEG-HOC-Val-Thr-Gnf-Ser-Asp-MOC PEG-HOC-Ala-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln-MOC PEG-FAM-Tyr-Tyr-Abu-BAD PEG-FAM-Val-Thr-Gnf-Ser-Asp-BAD PEG-FAM-Ala-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln-BAD PEG-HMC-Tyr-Tyr-Abu-CM3 PEG-HMC-Val-Thr-Gnf-Ser-Asp-CM3 PEG-HMC-Ala-Pro-Glu-Glu-Ile-Met-Asp-Arg-Gln-CM3

MW

Rt [min] enzyme

1221.3/1221.5 1416.4/1416.5 2036.2/2036.3 1375.4/1375.3 1570.6/1571.0 2050.2/a50.4 1300.4/1300.4 1495.6/1496.1 1959.1/1959.2

17.34 18.83 20.67 26.75 22.28 25.01 19.20 21.61 24.74

PR3 CG HNE PR3 CG HNE PR3 CG HNE

kcat [s−1] 9.7 3.1 5.1 3.8 2.7 6.2 7.8 7.2 1.3

± ± ± ± ± ± ± ± ±

1.5 0.4 1.3 1.2 0.3 0.9 0.7 2.1 0.1

KM [M] × 10−6 18.5 27.6 125.1 31.4 9.9 24.2 24.3 19.0 74.5

± ± ± ± ± ± ± ± ±

0.4 5.3 8.3 2.2 1.1 3.1 1.2 1.1 3.7

kcat/KM [M−1 × s−1] × 104 52.41 11.21 4.08 12.22 27.27 25.60 32.15 37.90 7.41

± ± ± ± ± ± ± ± ±

3.21 0.95 1.05 5.27 1.51 1.64 2.83 1.89 0.36

a

Where PEG = 2-(2-(2-aminoethoxy)ethoxy)acetic acid; HOC = Lys(Hoc)−OH, where Hoc = HOC 7 hydroxy-2-coumarin; MOC = Lys-(Moc)− OH, where Moc = 7 methoxy-coumarin; FAM = Lys(Fam)−OH, where Fam = 5(6)-fluorescein; BAD = Ala(Bad)−OH, where Bad = [benzo[b]acridin-12(5H)-on-2-yl; HMC = Lys(Hmc)−OH, where Hmc 7-hydroxy-4-methyl coumarin; CM3 = Orn(Cm3)−OH, where Cm3 = 3coumarin 343.

mixtures of substrates were incubated with activated and nonactivated human neutrophils (the amount varied from 3.5 × 104 to 7.1 × 105 cells). For all three substrates incubated with nonactivated cells, we observed no significant fluorescence increase (Figure 6C, Supporting Information). In contrast, for stimulated cells, a huge fluorescent boost was obtained when a mixture of three substrates (1, 6, and 8) was added (Figure 6C, Supporting Information). To find out if the fluorescence increase is an effect of NSP activity, an excess (100 nM) of PMSF (strong irreversible serine protease inhibitor) was added to the activated cells. As a result, we observed no fluorescence boost (not shown). This indicates that substrates are not nonspecifically processed by any other class of enzymes present in the investigated cells. We also preincubated the activated neutrophils (6 × 105 cells) with PR3 specific inhibitor,9 and no fluorescence increase characteristic for the PR3 substrate was observed (Figure 6D, Supporting Information). This showed that substrate 1 is selectively hydrolyzed only by PR3. Subsequently, we decided to test all of the substrates (1, 6, and 8) for their cell permeability properties. The 3 × 106 cells were incubated with 1, and after 5 and 20 min, 1 × 106 cells were removed, washed 3 times with PBS, and analyzed under a fluorescent microscope and with flow cytometry. A small amount of the incubated cells (5 μL) were removed for inspection. We observed an intense fluorescence visible inside the cell (see Figure 4). Results of the flow cytometry analysis indicated that the highest concentration of substrate 1 is achieved after 20 min of incubation with neutrophils (see Figure 5). The same procedure was repeated for substrates 6 and 8. The tested substrates efficiently penetrate the neutrophil membrane, albeit the fluorescence was predominantly located in the cytosol and not the nucleus. Since substrates are cell permeable and are proteolytically processed in pH that is close to the cytosol of the neutrophils, we decided to incubate substrates 1, 6, and 8 in neutrophil lysate in two buffers with different pH (7.5 and 5.5). As indicated in Figure 6, we observed a fluorescence increase during the time period in both pH systems. However, in pH 7.5, this phenomenon is sensitive to the presence of the neutrophil protease inhibitors (Figure 7, the azaPR39 was used to inhibit PR3), whereas in pH 5.5 it is not. We assume that in lower pH, the acidic proteolytic enzymes (cathepsins) are able to process the studied substrates. Also, there are differences in fluorescence increase for all substrates observed for the neutrophil lysate of both healthy donors and Wegener-diagnosed people. As can be seen in

Figure 3. The pH dependence of specificity parameters of selected substrates (1, 6, and 8).

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substrates can easily penetrate into the active site of the enzyme, and thus, they are proteolytically processed. Bearing in mind, all the above, we incubated the selected substrates with human serum. The results shown in Table 3 indicate that we were able to differentiate activity of the NSPs in serum of healthy people and patients with diagnosed diseases. The same results were obtained when a mixture of three substrates was used. Incubating the serum with PMSF followed by the addition of the substrate or mixture of the substrates resulted in no visible fluorescence increase (data not shown). In the case of serum that comes from patients with diagnosed Wegener disease, we observed a significant boost in the activity level of PR3 but not CG or HNE. However, the number of samples (12) did not allow us to draw more significant conclusions. With respect to analysis of sera (17 samples) of patients diagnosed with microscopic polyangiitis, we did observe an increase in all three enzymes; however, the activity of PR3 was not so strongly manifested as for WG patients (Table 3).

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DISCUSSION Nine new fluorescent substrates displaying FRET were designed and synthesized. Each compound contained a pair of fluorescent amino acids. Five out of six such derivatives were obtained by attachment of selected coumarin based fluorophores (Fam, Hoc, Moc, Cm3, Hmc) to the amino group of the side chain of basic amino acids (Lys or Orn). The remaining one (Bad) can be considered an L-alanine derivative substituted in the side chain by acridine moiety. These compounds were incorporated into the peptide chain of the substrates that were previously reported as selective substrates of NSPs. Finally, potent substrates of cathepsin G (peptide 8, kcat/kM = 37.9 × 104 M−1 × s−1), PR3 (peptide 1, kcat/kM = 52.4 × 104 M−1 × s−1), and HNE (peptide 6, kcat/kM = 37.9 × 104 M−1 × s−1) were obtained. Peptides 1 and 8 revealed higher specificity constants (kcat/kM) as compared to their parent compounds that were based on ABZ and MCA moieties.11,12,15 We observed a 3-fold increase for 1 and an over 2-fold increase for peptide 8. In the case of 6, a 2-fold reduction in specificity was observed. However, all the obtained peptides are cell permeable while their precursors are not. Such a feature could potentially be useful when monitoring proteinase activity inside the cell. As indicated by the results of the HPLC experiments followed by the MS analysis of fragments, all of the substrates incubated with proteinases are efficiently and selectively singlecleaved by the cognate protease. Within 1 h of incubation, there are no additional cleavages except for/apart from the P1−P1′ peptide bond. The same fact is observed upon monitoring of emission wavelength characteristics for the donor of fluorescence when an equimolar mixture of three substrates was incubated with one of the proteases (Figure 3). The energy transfer systems were designed on the basis of the spectral properties of the amino acid residues present at the C- and N-termini of the synthesized peptides. In the case of selected substrates (1, 6, and 8), both the three energy transfer systems (BAD-FAM), (MOC-HOC), and (HMC-CM3) and the quenching efficiency for each pair were determined. Three pairs display efficient energy transfer yielding from 50 to 40% due to excellent overlapping of the emission spectra of appropriate donor (BAD, MOC, HMC) and absorbance spectra of acceptors (FAM, HOC, CM3). The substrates

Figure 4. Fluorescent microscope images of the incubation of selected substrates with activated neutrophils: (A) HNE substrate (6), (B) CG substrate (8), (C) PR3 substrate (1).

Figure 6, the highest increase was observed for PR3. However, significantly increased activity of CG and HNE was recorded. Since NSP members are stored inside the granules of neutrophil and released upon its activation, we decided to test the level of those enzymes in human serum. According to the literature data,19−23 most of the NSP population are in complexes with its cognate inhibitors (antichymotrypsin, antitrypsin elafin, secretory leukocyte peptidase inhibitor (SLPI) or in nonspecific macroglobulin− enzyme complexes. Using the substrate excess and according to the Michealis-Menten kinetics, such noncovalent complexes could be disrupted. Additionally, in the macroglobulin− protease complex, the active site of the enzyme is masked to prevent digestion of large protein substrates. Low-molecular 7246



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Figure 5. Flow cytometry assay of neutrophils incubated with HNE substrate (6) in time periods: 0, 5, and 20 min.

Inflammation is a process that involves neutrophil recruitment and activation; thus, any of the substrates or their equimolar mixtures could serve as a marker of inflammation. Also, the samples from healthy people and those diagnosed with microscopic polyangiitis were examined for NSP activity. A significant increase in activity was observed for nonhealthy individuals. In the case of Wegener-diagnosed patients, there was a dramatic increase (10 times) in the activity of PR3. Also, such behavior was noticed when substrate mixtures were incubated with serum of Wegener-diagnosed patients. This correlates well with data from our previous research where we observed a huge discrepancy in PR3 activity for healthy and Wegener-diagnosed people.

Figure 6. Incubation of substrates 1, 6, and 8 with neutrophil lysates of healthy and Wegener-diagnosed people in two distinct pH solutions.

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CONCLUSIONS Three new selected FRET displaying substrates of HNE, PR3, and CG were synthesized and characterized. All the selected substrates displayed high values of specificity constants against target proteases, and each of the peptides was selectively hydrolyzed only by a cognate enzyme; this was confirmed by fluorometric and HPLC analyses. Additionally, the values of the kinetic parameters determined for substrate mixtures are very similar to those obtained for single compounds, indicating a lack of cross-activity among the investigated substrates. The same results were obtained when substrates were incubated with activated neutrophils. When enzymes or cells were treated with selective inhibitors of PR3 or inhibitors of serine proteases, neither PR3 nor NSP activity was observed. The obtained substrates were cell permeable and when incubated with neutrophils were quickly internalized into the cells. They also displayed significant activity when incubated with cell lysates which could indicate that they are processed within the neutrophil. Moreover, measurable activity of those proteases with the obtained substrates was detected in sera samples of healthy individuals, and a significant divergence was observed both in the case of people diagnosed with Wegener granulomacitosis and microscopic polyangiitis. We showed herein that using a three wavelength substrate system we are able to measure simultaneously three distinct activities of three neutrophil serine proteases in vitro and in vivo.

Figure 7. Effect of reversible PR3 inhibitor on proteolytic processing of substrate 1 in healthy and Wegener-diagnosed neutrophil lysates.

Table 3. NSP Activity in Serum of Healthy and Ill People disease (number of patients)

CGa

HNEa

PR3a

healthy individuals (28) Wegener disease (12) microscopic polyangiitis (17)

100 ± 30 122 ± 35 180 ± 41

100 ± 21 210 ± 42 270 ± 54

100 ± 26 1100 ± 210 380 ± 87

a

Activity is expressed as [%] of NSP activity determined in healthy individuals.

follow the pH patterns of NSP members and display a maximum specificity between pH 7 and 8. The three-substrate system was proved to be a good marker of neutrophil activation. For resting neutrophils, no significant fluorescence increases were observed when the discussed substrates were applied. In case of activated cells, a boost in fluorescence due to protease secretion was observed.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 7247



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by MNiSzW under grant IP2010 0484 70. REFERENCES

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