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Published ahead of print on December 23, 2003, doi:10.1165/rcmb.2003-0139OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 801-807, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0139OC

Design and Use of Highly Specific Substrates of Neutrophil Elastase and Proteinase 3

Brice Korkmaz, Sylvie Attucci, Thierry Moreau, Emmanuel Godat, Luiz Juliano and Francis Gauthier

INSERM U618, Protéases et Vectorisation Pulmonaires, University François Rabelais, Tours, France; and Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

Address correspondence to: Francis Gauthier, INSERM U618 "Proteases et Vectorisation Pulmonaires," University François Rabelais, 2 bis Bd Tonnellé, 37032 Tours Cedex, France. E-mail: gauthier{at}univ-tours.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have exploited differences in the structures of S2' subsites of proteinase 3 (Pr3) and human neutrophil elastase (HNE) to prepare new fluorogenic substrates specific for each of these proteases. The positively charged residue at position 143 in Pr3 prevents it from accommodating an arginyl residue at S2' and improves the binding of P2' aspartyl-containing substrates, as judged by the decreased Km. As a result, the kcat/Km for Abz-VADCADQ-EDDnp is over 500 times greater for Pr3 than for HNE, and that for Abz-APEEIMRRQ-EDDnp is over 500 times greater for HNE than for Pr3. This allows each protease activity to be measured in the presence of a large excess of the other, as might occur in vivo. Placing a prolyl residue in position P2' greatly impaired substrate binding to both HNE and Pr3, which further emphasizes the importance of S' subsites in these proteases. HNE and Pr3 activities were measured with these substrates at the surface of fixed polymorphonuclear leukocytes (PMNs) before and after activation. This demonstrated that their active site remains accessible when they are exposed to the cell surface. Both membrane-bound proteases were strongly inhibited by low Mr serine protease inhibitors, but only partially by inhibitors of larger Mr such as {alpha}1-protease inhibitor, the main physiologic inhibitor in lung secretions. Most of membrane-bound HNE and Pr3 can be released from the membrane surface of fixed cells by a buffer containing detergent, suggesting that hydrophobic interactions are involved in membrane binding.

Abbreviations: {alpha}1-protease inhibitor, {alpha}1-PI • ortho–aminobenzoic acid, Abz • 7-amino-4-trifluoromethyl-coumarin, AFC • p-aminophenylmercuric acetate, APMA • bronchoalveolar lavage, BAL • chloromethyl ketone, CMK • cytokine-response modifier A, CrmA • N-(2,4-dinitrophenyl) ethylenediamine, EDDnp • L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanido)butane, E64 • fluorescein isothiocyanate, FITC • 9-fluoroenylmethoxycarbonyl, FMOC • human neutrophil elastase, HNE • high-performance liquid chromatography, HPLC • methoxysuccinyl, MeO-Suc- • turkey ovomucoid inhibitor domain 3, OMT3 • plasminogen activator inhibitor 1, PAI-1 • phosphate-buffered saline, PBS • polymorphonuclear leukocyte, PMN • phenylmethylsulfonyl fluoride, PMSF • proteinase 3, Pr3 • nitrotyrosine, Y(NO2)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Serine proteases from the azurophil granules of human polymorphonuclear neutrophils (PMNs) are directly and indirectly involved in the pathophysiology of acute lung injury because of their capacity to degrade most components of the extracellular matrix (1–4) and trigger the release of proinflammatory cytokines and chemoattractants (5–7). Knowledge of the roles of these serine proteases, human neutrophil elastase (HNE), proteinase 3 (Pr3), and cathepsin G, may help show how lung inflammatory disorders are triggered, spread, and become chronic. The proteases could be targets for treatment with specific inhibitors that reduce their proteolytic activity (8). But before any such application, we need to know the activities of each serine protease, especially in lung secretions, where they may be present together with other endogeneous or exogeneous (bacterial) proteases (8). As a first step in that direction, we recently developed new synthetic substrates that are sensitive enough to measure the activities of all three proteases at nanomolar concentrations, when they are present in similar amounts in biological fluids or at the surface of cells (9, 10). This should occur normally, as HNE, Pr3, and cathepsin G are all stored in similar concentrations in the primary granules of PMNs, and are released at the same time upon PMN activation and degranulation (8, 11, 12). However, recent data suggest that this is not always the case and that their concentrations may differ depending on their binding to the cell membrane (10, 11), their sensitivity to endogenous inhibitors at inflammatory sites (8), their specificity toward peptide or protein substrates (13), and on their regulation by other components such as DNA (14), autoantibodies (15), heparin (16–18), and bacterial proteases (19). This could also depend on alternative storage conditions within the PMNs and interindividual distribution at the PMN surface as described for Pr3 (20, 21). The relative amounts of these proteases in biological fluids, such as bronchoalveolar lavage (BAL) fluids, has not been carefully investigated until now due to the lack of efficient, sensitive substrates. This lack has seriously impaired studies on the specific function of each protease during acute lung diseases (11). As the relative concentrations of active HNE and Pr3 in biological fluids may differ significantly, more specific substrates are required to measure the activities of low concentrations of HNE or Pr3 in the presence of higher concentrations of the other.

Though HNE and Pr3 are very closely related structurally and functionally, crystallographic data clearly show that the prime side of the binding cleft of Pr3, which covers subsites from S1' to S3', is more restrictive and definitely more polar than that of HNE (22). We have therefore produced new substrates based on the sequences of those reported previously (9, 10) by changing the lengths and amino acid compositions on the prime side of the substrates. We also compared the specificities of PMN membrane–bound and free proteases on the newly developed substrates, because we have recently shown that the proteolytic activity of elastase-like proteases in BAL fluids from patients with mild inflammatory pulmonary disease is essentially membrane-bound (Hazouard and coworkers, unpublished results).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
HNE (EC 3.4.21.37), proteinase 3 (EC 3.4.21.76), {alpha}1-antichymotrypsin, and {alpha}1-protease inhibitor ({alpha}1-PI) were obtained from Athens Research and Technology (Athens, GA). Trypsin (EC 3.4.21.37) was from Sigma (St. Quentin Fallavier, France) and cathepsin G (3.4.21.20) from ICN Pharmaceuticals (Orsay, France). proMMP8 (EC 3.4.24.34) and proMMP9 (EC 3.4.24.35) and their respective synthetic substrates (DNP-PLAYWAR-OH and DNP-PLGMWSR-OH) were from Calbiochem (France Biochem, Meudon, France). MeO-Suc-AAPV-AFC was from Enzyme System Products (Livermore, CA) and MeO-Suc-AAPA-CMK from Bachem (Weil am Rhein, Germany). E64, Igepal CA-630, A23187, and APMA were from Sigma. N,N-dimethylformamide and acetonitrile were from Merck (Merck Eurolab, Strasbourg, France), C18 cartridges for reverse-phase chromatography were from VWR International S.A.S (Strasbourg, France). Polymorphprep and Lymphoprep were from Nycomed Pharma (Oslo, Norway). Monoclonal mouse IgG1-FITC and CD63-FITC antibodies were from Beckman Coulter France (Roissy, France). All other reagents were of analytical grade.

Isolation of Blood PMNs
Human PMNs were purified from 8-ml samples of peripheral blood collected from healthy volunteers into EDTA-containing tubes as previously reported (9) with the following modifications. The PMN pellet recovered after lysing the erythrocytes was washed twice in phosphate-buffered saline (PBS) containing 4 mM EGTA and 1% bovine serum albumin. Purified PMNs were kept at room temperature in this buffer with gentle shaking, then washed in PBS supplemented with 4 mM EGTA until the enzymes were assayed. Flow cytometry was performed on a Coulter Epics Elite ESP flow cytometer equipped with a 488-nm argon laser. The forward and side scatter of each sample were measured for at least 10,000 events. Samples contained more than 99% PMNs, no monocytes, and less than 1% lymphocytes. The absence of CD63 at the surface of freshly prepared PMNs was checked by incubating 5 x 105 PMNs with 20 µl monoclonal anti-IgG1-FITC or anti-CD63-FITC antibodies for 20 min at room temperature. The mixtures were centrifuged at 500 x g for 5 min and the resulting pellets washed twice in PBS and resuspended in PBS for flow cytometry analysis. Cell viability was checked by trypan blue exclusion.

PMN Activation and Fixation
The PMNs were washed in PBS, suspended at ~ 3 x 106 cells/µl in PBS containing 1 mM CaCl2 and 1 mM MgCl2, and incubated at 37°C for 15 min with A23187 (1 µM final). The activated PMNs were fixed by incubation for 3 min on ice with 3% (wt/vol) paraformaldehyde and 0.25% (vol/vol) glutaraldehyde, as reported by Owen and coworkers (12). The PMNs were washed twice with PBS-EGTA, suspended at ~ 3 x 106 cells/µl in the same buffer, and used for enzyme assays.

Design and Synthesis of Quenched Fluorescent Substrates
All Fmoc-protected amino-acids were of the L-configuration, and were purchased from Advanced Chemtech (Louisville, KY), Applied Biosystems (Warrington, UK), and Neosystem (Strasbourg, France). Abz-peptidyl-EDDnp fluorogenic substrates were prepared by solid-phase synthesis with Fmoc methodology using a multiple automated peptide synthesizer (PSSM-8; Shimadzu Co., Kyoto, Japan) (23). Glutamine was the C-terminal residue in all peptides, due to a requirement of the synthesis strategy (23).

Abz-peptidyl Y(NO2) fluorogenic substrates were synthesized by Fmoc chemistry on an automated Multiple Peptide Synthesis System (Pioneer; Applied Biosystems, Foster City, CA) using an Fmoc-PAL-PEG-PS (5-(4-(9-Fluoroenylmemethoxycarbonyl) aminophylline-3, 5-dimethoxyphenoxy) valeric acid-polyethylene glycol-polystyrene) amide resin (Warrington, UK). Substrates were purified by semipreparative reverse-phase chromatography using a 50-min linear (0–100%) gradient of acetonitrile in 0.1% trifluoroacetic acid and checked for homogenity by analytic RP-HPLC on a C18 column and by MALDI-TOF mass spectrometry (TofSpec-E; Micromass, Manchester, UK) or peptide sequencing (Applied Biosystems Procise Sequencer) with the chemicals and program recommended by the manufacturer.

Stock substrate solutions (2–5 mM) were prepared in 30% (vol/vol) N,N-dimethylformamide and diluted to 0.5 mM with 50 mM Hepes buffer pH 7.4. Cys-containing substrate stock solutions were supplemented with 10 mM dithiothreitol.

Enzyme Assays
Free HNE and Pr3 were measured in 50 mM Hepes, pH 7.4, 150 mM NaCl, supplemented with 0.05% Igepal CA-630 (vol/vol) to take into account the great propensity of these proteases to stick to plastic and glass surfaces when in dilute solution. The activities of free proteases were also measured in the detergent-free buffer used for membrane-bound proteases to allow comparison between free and membrane-bound activities. Free HNE and Pr3 were titrated with {alpha}1-PI, the titer of which had been determined using bovine trypsin titrated with p-nitrophenyl-p'-guanidinobenzoate (24). Whereas a 1:1 stoichiometry was used for HNE, that for the Pr3/inhibitor complex was 1:1.3 to allow for partial {alpha}1-PI degradation via the substrate pathway (25). Membrane-bound active proteases were quantified by comparing the rate of hydrolysis of their specific substrate to that of titrated proteases working in the same experimental conditions. Trypsin was prepared as a 2 x 10–4 M stock solution in 100 mM Tris/HCl buffer pH 8:50 mM CaCl2, then used in the same buffer as HNE and Pr3. The hydrolysis of Abz-peptidyl-EDDnp and Abz-peptidyl-Y(NO2) substrates was followed by measuring the fluorescence at {lambda}ex = 320 nm and {lambda}em = 420 nm in a Hitachi F-2000 spectrofluorometer. The system was standardized using Abz-FR-OH prepared by the total tryptic hydrolysis of an Abz-FR-pNA solution, and its concentration was determined from the absorbance at 410 nm, assuming E410 nm = 8,800 M–1 cm–1 for p-nitroanilide. Concentrations of Abz-peptidyl-EDDnp substrate were determined by measuring the absorbance at 365 nm, using E365 nm = 17,300 M–1 cm–1 for EDDnp.

Specificity constants (kcat/Km) were determined under first-order conditions using a substrate concentration far below the Km (max 1 µM). Final enzyme concentrations were 10–50 nM for HNE and Pr3, 100 nM for cathepsin G, and 5–10 nM for trypsin. Under these conditions, the Michaelis-Menten equation is reduced to: v = kobs.S, where kobs = Vm/Km. Integrating this equation over time gives ln (S) = -kobs.t + ln(S)o, with (S)o and (S) being the substrate concentrations at time 0 and time t, respectively. Because Vm = kcat. (E)t, where (E)t is the final enzyme concentration, dividing kobs by (E)t gives the kcat/Km ratio. The kobs for the first-order substrate hydrolysis was calculated by fitting experimental data to the first-order equation using Enzfitter software (Elsevier Science Publishers, Amsterdam, The Netherlands). Km values were determined by measuring hydrolysis rates at five substrate concentrations from 1–8 µM.

proMMP8 and proMMP9 were activated by incubating them with 1 mM freshly prepared APMA at 37°C for 150 min in 50 mM Tris/HCl buffer pH 7.0, 200 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.05% Brij 35, 0.05% NaN3. They were then desalted and assayed at a final concentration of 5 x 10–8 M in 50 mM Tris/Tricine pH 7.5, 200 mM NaCl, 10 mM CaCl2, 50 µM ZnCl2.

Enzyme Activity at the Surface of PMNs
Fixed, unactivated, and activated PMNs (2.5 x 105–106 cells), or purified proteases used as controls, were incubated with 10 µM fluorogenic substrate in polypropylene microplate wells (Hard-Shell Thin-Wall Microplates; MJ Research, Waltham, MA) at 37°C in a detergent-free activity buffer (50 mM PBS, pH 7.4) to avoid the release of protease during kinetic measurements. The fluorescence was recorded continuously using a microplate fluorescence reader (Spectra Max Gemini; Molecular Devices, St. Grégoire, France) under continuous stirring. The release of membrane-bound proteases into PBS was checked by incubating cells for 2 h in this buffer, and measuring the peptidase activities in supernatants cleared of cells by centrifugation for 5 min at 500 x g. PMSF was freshly prepared and used at a final concentration of 1 mM. It was incubated with ~ 106 PMNs, or purified HNE or Pr3 (3 x 10–9 M final) for 20 min at 37°C. Cells were collected by centrifugation for 10 min at 500 x g and suspended in PBS. The enzyme activities in the pellet were measured in 50 mM Hepes, pH 7.4, 150 mM NaCl, 0.05% Igepal 630. {alpha}1-Pi (1 µM final) was incubated with 2.5 x 105–106 PMNs, or purified HNE or Pr3 (0.5 to 3 x 10–9 M final) in PBS for 60 min at 37°C before recording enzyme activities with an appropriate substrate. E64 was used at a final concentration of 10 µM and EDTA at 1 mM.

Chromatography and Analysis of Peptide Products
Fluorogenic substrates (4–8 µM final) were incubated with HNE or Pr3 (10–100 nM) in reaction buffer at 37°C. The reaction was stopped by adding 4 vols absolute ethanol and incubation for 15 min on ice. Precipitated protein was removed by centrifugation at 13,000 x g for 10 min. The supernatant containing the hydrolysis products was dried under vacuum and dissolved in 200 µl 0.01% trifluoroacetic acid (vol/vol). Hydrolysis fragments were purified by reverse phase chromatography on a C18 column (2.1 mm x 30 mm) using a Thermo Separation Product (TSP, Les Ulis, France) P200 pump and a Spectrasystem UV3000 detector (TSP). They were eluted at 0.3 ml/min with a linear (0–60%, vol/vol) gradient of acetonitrile in 0.01% trifluoroacetic acid for 20 min. Eluted peaks were monitored at three wavelengths (220, 320, and 360 nm) simultaneously, which allowed the direct identification of EDDnp-containing peptides prior to sequencing. Cleavage sites were identified by N-terminal sequencing.

Electrostatic Potential Calculations
The electrostatic potentials of HNE and Pr3 were calculated using the Poisson-Boltzmann method implemented in the Delphi module of InsightII (Accelrys Inc., San Diego, CA) using formal charges at pH 7.4 (arginine, lysine, N-terminus, +1; glutamate, aspartate, and the C-terminus, –1; and histidine, neutral), an ionic strength in the aqueous environment of 0.15 M, dielectric constants of 2 for the interior and 80 for the exterior of the protein, and a temperature of 300 K.

The X-ray coordinates of HNE were extracted from the complex formed by HNE with the turkey ovomucoid inhibitor OMT3 (PDB accession no. 1ppf). The structure of Pr3 (PDB accession no. 1fuj) was superimposed on that of HNE to visualize both molecules with the same orientation. The location of subsites S2 to S3' in the active site of each protease was inferred from the position of the side-chains of the corresponding P2-P3' residues of OMT3 complexed with HNE. The solvent-accessible molecular surface was calculated and colored according to the electrostatic potential using Insight II (Accelrys Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preparing a Specific Substrate for Pr3
The crystallographic data reported by Fujinaga and coworkers show that the interaction sites of Pr3 and HNE extend on both sides of the reactive bond, but that the S' subsites of Pr3 are more restrictive than are those of HNE (22). Crystallographic data also show that the S2' subsite of Pr3 is more polar than that of HNE, because of the replacement of Leu by Arg at position 143 and of Ile by Pro at position 151 (22). A negatively charged residue at P2' in a Pr3 substrate would therefore improve its interaction with the Pr3 S'2 subsite, whereas a positively charged residue at that position would not. We therefore inserted charged or polar residues at P2' in the CrmA-derived substrate Abz-VADCAQ-EDDnp (substrate 1), which is cleaved at the unusual C–A bond by Pr3 (10).

We also studied the possible influence of EDDnp on the rate of hydrolysis by using Y(NO2) as a quencher, because of the importance of the S' site beyond S2' in Pr3. This substitution also permitted the deletion of the Gln residue that is routinely introduced in the penultimate position of Abz-peptidyl-EDDnp substrates because of the synthesis methods used, and which may sometimes favor cleavage by HNE when present at P1' (10).

Shortening the substrate P' side (Table 1, substrate 2) or introducing an Arg into P2' (substrate 3) greatly reduced the sensitivity to Pr3, but made this an excellent substrate for trypsin (Table 1). The presence of an aspartyl residue at P2' (substrate 4) not only avoided this drawback, it also significantly improved the sensitivity to Pr3, with a kcat/Km greater than 6 x 105 M–1s–1. It was significantly less sensitive to HNE than the lead substrate (substrate 1). This increased specificity was mainly due to the Km (Km = 6.5 ± 1.1 µM) being halved. The presence of an additional Arg residue at P3' in substrate 6 (Abz-VADCADR-Y(NO2)) increased P' side polarity without changing the kcat/Km for Pr3, but this made it totally resistant to HNE (Table 1). However, substrate 6 was cleaved slowly by trypsin because of the Arg residue. The quenching group Y(NO2) was replaced by Q-EDDnp in substrate 7 (Abz-VADCADQ-EDDnp) to take into account the possible influence of the quencher on substrate binding. The kcat/Km was similar to that obtained with substrate 4 (Abz-VADCAD-Y(NO2)), but it was cleaved more slowly by HNE. These results demonstrate the importance of the S2' subsite in Pr3 for substrate hydrolysis, a conclusion reinforced by the observation that a single change from Asp to Pro at that position reduced the rate of cleavage ~ 600-fold (Table 1). Pr3 still cleaved Abz-VADCAD-containing substrates at the same C–A bond, as demonstrated by HPLC and N-terminal sequencing of the EDDnp-containing peptide (not shown).


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  		TABLE 1. Influence of P2' residues on the specificity constants kCAT/KM for the hydrolysis of fluorogenic substrates by proteinase 3

 
Preparing a Specific Substrate for HNE
The best HNE substrate we have prepared before from the reactive loop sequence of the PAI-1 serpin, (Abz-APEEIMDRQ-EDDnp; Table 2, substrate 8) (10) is cleaved at the I-M bond, and therefore contains an Asp at P2', as do the Pr3 substrates described above. This is not incompatible with the presence of a Leu residue in the S2' subsite of HNE, especially if the S2' subsite of HNE is not of prime importance for substrate binding (26).


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  		TABLE 2. Influence of P2' residues on the specificity constants kCAT/KM for the hydrolysis of fluorogenic substrates by human leucocyte elastase

 
Substrate 8 was also cleaved at the same site by Pr3, though at a far slower, but still significant, rate (Table 2). As reported above, an Arg residue at P2' in the HNE substrate probably impaired hydrolysis by Pr3, but possibly not by HNE. We checked this by replacing Asp by Arg at P2' in substrate 8. We also synthesized a series of related substrates with polar residues at P3' to check the influence of positively or negatively charged groups at that position. Finally, we checked the influence of the quenching group by replacing the EDDnp group in the lead substrate by a nitrotyrosyl group.

The results (Table 2) show that an Arg residue can be accommodated by the HNE S2' subsite as well as an Asp residue, as deduced from the similar specificity constants (kcat/Km). N-terminal sequencing of the EDDnp-containing fragment showed that the cleavage site was still the Ile-Met bond. As expected, Pr 3 hardly hydrolysed this substrate; the kcat/Km was about ten times lower than that for the Asp-containing substrate. A Pro residue at P2' strongly impaired hydrolysis by HNE, as observed for Pr3. Changing residues in P3' has little influence on hydrolysis by HNE, but both HNE and Pr3 cleaved substrates with an Arg at that position more readily. The Arg-containing substrates 8–10 were cleaved at a significant rate by trypsin, and cleavages at the Arg–Gln bond were identified by N-terminal sequencing. Though two Arg residues are present in substrate 9, no cleavage at the Arg–Arg bond was identified. Finally, substituting a nitrotyrosyl group for Gln-EDDnp resulted in a 10-fold decrease in kcat/Km, suggesting that the quencher group helps stabilize the substrate at the protease surface (Table 2).

Competition between HNE and Pr3 for Abz-peptidyl-EDDnp Substrates
The great specificity of VADCAD-containing peptide substrates for Pr3, and that of the APEEIMRRQ sequence for HNE should allow measurements of low concentrations of either protease in the presence of larger amounts of the other, as may occur in vivo. This was checked by mixing Pr3 (10–9 M final) with up to a 50-fold molar excess of HNE and comparing the rates of cleavage of the Pr3 substrate 7 (Abz-VADCADQ-EDDnp) with that of Pr3 alone. Conversely, we mixed HNE (10–9 M final) with up to a 50-fold molar excess of Pr3 and compared the rates of cleavage of the HNE substrate 9 (Abz-APEEIMRRQ-EDDnp) with that of HNE alone. The same experiments were also done using the lead substrates (substrates 1 and 8). The rates of cleavage by HNE and Pr3 in the presence or absence of excess of the other protease were identical using the new substrates described here, although this was not so using lead substrates under the same conditions (Figure 1). We conclude that HNE and Pr3 activities can be measured in whole biological fluids whatever their relative concentrations, provided that no other yet unidentified protease in the sample can cleave these substrates at a significant rate.



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  		Figure 1. Protease concentration–dependent hydrolysis of Pr3 and HNE substrates. (A) Pr3 substrates (5 mM final) were hydrolyzed by Pr3 (10–9 M) used as a control, and by HNE (10–9 M and 5 x 10–8 M) with or without Pr3 (10–9 M). (B) HNE substrates (5 mM final) were hydrolyzed by HNE (10–9 M) used as a control, and by Pr3 (10–9 M and 5 x 10–8 M) in the presence or absence of HNE (10–9 M).

 
Electrostatic Surface Potential of Pr3 and HNE
The position of Arg 143 at the S2' site of Pr3 (Figure 2) supports the idea that the great specificity of the substrates described here probably depends on the replacement of Leu in HNE by Arg at position 143 in Pr3. The S' subsites at the protease surface were deduced from the positioning of P' residues in the turkey inhibitor OMT3 in complex with HNE. Examination of the electrostatic surface potentials of HNE and Pr3 also indicated how HNE and Pr3 accommodate negatively charged residues on their unprime side (Figure 2).



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  		Figure 2. Electrostatic potential of PMN elastase and Pr3. (A) Solvent-accessible surface of HNE and Pr3 colored according to its positive (dark blue) and negative (red) electrostratic potential. The boxed regions correpond to the active site of the enzymes. The Arg143 residue in Pr3 lining the S2' pocket of the active site is indicated. The contouring level of electrostatic potential is –5 kT/e (red) and +5 kT/e (blue). (B) Close view of the active site region of HNE and Pr3 with the reactive site loop of the inhibitor OMT3 complexed with HNE (left). The putative OMT3–Pr3 complex (right) was obtained by superimposing Pr3 onto the OMT3-HNE complex. Side-chains of the inhibitor residues (P2 to P3' residues) interacting with the enzyme active site occupy the corresponding binding pockets (S2 to S3'). Arg143 of Pr3 (Leu in HNE) clearly contributes to forming a polar S2' pocket in Pr3 but not in HNE, thus directing the P2' specificity of Pr3 toward acidic residues.

 
Measuring HNE and Pr3 Activities at the PMN Surface
There is now considerable evidence that serine proteases from azurophil granules are expressed at the surface of activated neutrophils, where their proteolytic activity might be of major biological relevance (11, 12, 27, 28). In the absence of specific substrates, only indirect methods based on the differing sensitivities of Pr3 and HNE to inhibitors have enabled assessment of the relative activities of these two proteases (29). The use of substrates such as those described here should circumvent this problem. Pr3 and HNE peptidase activities were measured in a detergent-free buffer, incubating 2.5 x 105–106 nonactivated and 105–5 x 105 activated fixed PMNs with substrates 7 and 8, respectively. The amounts of proteases at the surface of fixed PMNs were estimated to be 0.01–0.05 pg/cell for both HNE and Pr3, based on the rate of hydrolysis by titrated free HNE and Pr3 measured in the same buffer. This amount was increased by a factor of 3–5 for both proteases when the PMNs were activated by the calcium ionophore A23187. Each substrate was cleaved at a single site, the C–A bond (Pr3) and the I–M bond (for HNE), by comparing the HPLC profiles of hydrolysis products with those obtained using purified proteases (10). Incubation of the PMNs in the optimized HNE and Pr3 activity buffer that contains 0.05% Igepal-630 (10) to measure activities showed that Pr3 and to a lesser extent HNE were rapidly released from cell surfaces, though the integrity of fixed PMNs was preserved. Therefore free, rather than membrane-bound, activities were measured under these conditions, which resulted in 3- to 8-fold increases in the amounts of HNE and Pr3 detected when using the detergent-containing activity buffer. This could be because peptidyl substrates with intramolecularly quenched fluorescence have limited access to the active site of membrane-bound proteases.

Substrate and Inhibitor Access to Membrane-Bound Proteases
Because the peptidase activity measured at the cell surface was significantly lower than that released by the detergent-containing buffer, we checked to determine whether the negative charges of the HNE and Pr3 substrates developed here might impair access to the negatively charged membrane surface. We compared the rates at which most of HNE substrates in Table 2 and an uncharged HNE substrate (MeO-Suc-AAPV-AFC), used as a control, were cleaved by purified HNE and by activated, fixed PMNs. The net charge of the HNE substrates was –4 to 0, bearing in mind that the Abz group (pK = 4.3) is not charged at the experimental pH. The cleavage rate ratios (free protease/membrane-bound protease) were the same, whatever the net charge of the substrate. This suggests that the access of negatively charged HNE substrates to the cell surface is not significantly impaired by a charge effect. Because HNE and Pr3 are at the surface of both unactivated and activated PMNs, and because their activities were selectively measured using low Mr peptidyl substrates, we checked to see how these membrane-bound proteases are controled by protease inhibitors. PMSF, a broad spectrum low Mr serine protease inhibitor, totally inhibited the HNE and Pr3 bound to the membranes of both unactivated and activated PMNs within minutes, indicating complete access of low Mr inhibitors to the active sites of both proteases. The same result was obtained with HNE using the irreversible chloromethyl ketone inhibitor MeO-Suc-AAPA-CMK, a potent HNE inhibitor that inhibits Pr3 poorly. Unlike low Mr inhibitors, the physiological inhibitor {alpha}1-PI, used at a final concentration of 10–6 M, inhibited only 50–70% of membrane-bound HNE and Pr3, after incubation with 106 PMNs for 1 h. Similarly, a cocktail of cysteine and metalloprotease inhibitors (E64 + EDTA) had no effect on the rate of hydrolysis of HNE and Pr3 substrates by membrane-bound proteases.

We also checked that purified MMP8 and MMP9, the two main metalloproteases secreted by PMNs and possibly activated by HNE and cathepsin G (30, 31), did not cleave HNE and Pr3 substrates, even when assayed in appropriate MMP activity buffer.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
All the substrates used until recently to assay Pr3 were better hydrolyzed by HNE (3, 11, 20), so that the Pr3 in biological fluids or at cell surfaces could only be quantified by indirect methods. The biological relevance of Pr3 as a proteolytic enzyme was therefore difficult to assess, even though Pr3 has been shown to be involved in several pathophysiologic situations, such as Wegener granulomatosis (32), acute and chronic myeloid leukemia (33), cell proliferation (34), and apoptosis involving its C-terminal domain (35). We have recently demonstrated that HNE and Pr3 have different substrate specificities, and this has been confirmed by others (36). These different specificities depend greatly on S'–P' interactions, which explains why peptidyl substrates with intramolecularly quenched fluorescence that are cleaved within their peptidyl moiety to release fluorescence were used to reach this conclusion. The HNE and Pr3 substrates we recently prepared (10) allow almost specific measurements of each protease in a biological fluid when they are present in similar amounts. However their extracellular concentrations may differ significantly in time and space even though they are released simultaneously and in similar amounts from activated PMN primary granules. This depends on their attachment to membranes (11, 12), their sensitivity to natural protease inhibitors (8) and their interactions with a variety of compounds at inflammatory sites (13–19, 37). Differences in the secretion and cell surface exposure of proteases may also be due to alternative storage in secretory vesicles or to genetic factors, as shown for Pr3 (20). We took advantage of the differing structures of the S2' sites of HNE and Pr3 to raise more specific substrates that allow HNE and Pr3 to be quantified specifically in BAL fluids or sputum, whatever their relative concentrations. An Arg residue at position 143 of Pr3 prevents the protease from accomodating a positively charged Arg residue at S2', whereas this is well accepted by HNE. The drawback of this strategy, however, is that it introduces a cleavage site for trypsin-like proteases. But we found no cleavage at Arg residues in HNE substrates using BAL fluids, sputum, or purified PMNs. We also showed the influence of the length of the substrate P' moiety on the specificity constants, and the deleterious effect of a Pro at P2', which prevents almost all hydrolysis by either protease. The demonstration that highly specific Pr3 substrates can be obtained also reinforces the fact that the proteolytic activity of Pr3 is of physiologic relevance.

Low concentrations of HNE, Pr3, and cat G have been demonstrated at the surface of unstimulated human PMNs by immunofluorescence and immunogold staining (11, 12, 20). PMNs degranulate when activated by cytokines and chemoattractants, releasing large amounts of these proteases from their intracellular stores, part of which remains attached to membranes. The activity of membrane-bound proteases has been reported only with membrane fractions obtained after cell lysis (38), or using exogenous proteases that were bound to fixed PMNs (11, 12), but no report to date has demonstrated that membrane-bound endogenous Pr3 and HNE are catalytically active at the surface of whole resting or activated cells. We previously measured HNE and Pr3 activities by testing whole resting cells in protease activity buffer containing 0.05% Igepal-CA630 (10). But the presence of detergent significantly enhances the rate of hydrolysis and causes the rapid release of Pr3 and HNE from the membrane of fixed cells. Free, rather than membrane-bound, proteases are therefore measured under these conditions. Using a detergent-free buffer and fixed cells (fixed with paraformaldehyde/glutaraldehyde) maintains cell integrity, avoids any leakage of endogenous protease, and does not alter free HNE and Pr3 catalytic activities (12). We have now used this system to demonstrate that HNE and Pr3 are present as active proteases at the surface of unstimulated PMNs. This also allowed us to estimate the amount of active protease at the PMN surface. We find that the amount of active HNE and Pr3 bound to the surface of activated PMNs compares well with that reported by others (12). These membrane-bound proteases are fully inhibited by low Mr inhibitors, but far less efficiently by higher Mr protein inhibitors such as {alpha}1-PI, which agrees with the results reported by Owen and Campbell using exogenous proteases bound to fixed PMN (11, 12).

Pr3, and to a lesser extent HNE, are readily released from PMN membranes by low concentrations of detergent, which means that they are not simply linked through electrostatic interactions, as we and others have reported (10, 11). Other interactions, probably hydrophobic, also are involved in their binding to PMN membranes. Hydrophobic and covalent interactions have been reported for the binding of Pr3 to PMNs (20, 39). Cathepsin G, which is located with HNE and Pr3 within azurophil granules and is also at the PMN surface, is not released from the membrane by the detergent. We have also shown that membrane-bound cathepsin G hydrolyzes a negatively charged substrate less efficiently that a neutral substrate, unlike free cathepsin G (9). This indicates that the three neutrophil serine proteases are bound differently to membrane surfaces. We are currently investigating this in our laboratory, because of the putative physiologic importance of membrane-bound serine protease activity during inflammation.


   Acknowledgments
 
The authors thank Michèle Brillard-Bourdet, Catherine Girardin, and Alexandra Lafitte for technical assistance; Marie Lise Jourdan (INSERM E 0211) for flow cytometry analyses; Owen Parkes for editing the English text; and Joseph Bieth (INSERM U 392) for critically reading the manuscript. This work was supported in France by Vaincre la Mucoviscidose, Association pour la Recherche contre le Cancer (ARC), Biotechnocentre and Région Centre, and in Brazil by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PADCT).


   Footnotes
 
The nomenclature used for the individual amino acid residues (e.g. P1, P2, and so on) of a substrate and corresponding residues of the enzyme subsites (e.g. S1, S2, and so on) is that of Schechter and Berger (40).

Received in original form April 17, 2003

Received in final form December 15, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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