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Inhibition of Proteinase 3 by ₁-Antitrypsin In Vitro Predicts Very Fast Inhibition In Vivo

Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, Illkirch, France

Address correspondence to: Joseph G. Bieth, INSERM U 392, Faculté de Pharmacie, 74 route du Rhin, F-67400 Illkirch, France. E-mail: jgbieth{at}aspirine.u-strasbg.fr

	Abstract

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Neutrophil proteinase 3 (Pr3) cleaves elastin and other matrix proteins, and is thought to cause lung tissue destruction in emphysema and cystic fibrosis. Its deleterious action is theoretically prevented by ₁-antitrypsin, a serpin present in lung secretions. We have evaluated the anti-Pr3 activity of this inhibitor to decide whether it may play a physiologic proteolysis-preventing function in vivo. We show that (i) the oxidized inhibitor does not inhibit Pr3; (ii) the inhibitor competes favorably with elastin for the binding of Pr3, but is less efficient for inhibiting elastin-bound proteinase than for complexing free enzyme; and (iii) the inhibition takes place in at least two steps: the enzyme and the inhibitor first form a high-affinity reversible inhibitory complex EI* with an equilibrium dissociation constant K^*_i of 38 nM; EI* subsequently transforms into an irreversible complex EI with a first-order rate constant k₂ of 0.04 s^-1. Because the ₁-antitrypsin concentration in the epithelial lining fluid is much higher than K^*_i, any Pr3 molecule released from neutrophils will be taken up as an EI* complex within much less than 1 s, indicating very efficient inhibition in vivo.

Abbreviations: ₁-antitrypsin, 1-AT • ₁-proteinase inhibitor, ₁PI • human neutrophil proteinase 3, Pr3 • remazol-brilliant-blue-labeled bovine neck ligamentum elastin, RBB-elastin • methoxysuccinyl-lysine-(2-picolinoyl)-alanine-proline-valine-thiobenzyl ester, TBE

	Introduction

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The azurophilic granules of polymorphonuclear neutrophils contain three serine proteinases: elastase, cathepsin G, and proteinase 3 (Pr3), which participate in lysosomal bacterial digestion and neutrophil migration through the extracellular matrix at sites of inflammation. These enzymes are 30-kD glycoproteins that belong to the chymotrypsin family of serine proteinases. Pr3 is the most recently discovered, the most difficult to isolate, and hence, the less well studied proteinase of the three. It is identical to three independently-discovered proteins: (i) myeloblastin, which regulates the growth and differentiation of leukemic cells; (ii) p29b, which has microbicidal activity; and (iii) the target antigen of antineutrophil cytoplasmic autoantibodies detected in patients with Wegner's granulomatosis (1). Pr3 cleaves extracellular matrix proteins including elastin, type IV collagen, fibronectin, laminin, and vitronectin (2, 3).

Pr3 may be released from neutrophils during frustrated phagocytosis, cytokine-induced cell activation, or cell death (4). Lung tissue is theoretically protected against its deleterious action by a number of proteinase inhibitors including ₁ proteinase inhibitor (₁PI) (3), monocyte/neutrophil elastase inhibitor (5), and elafin (6). The lung secretory leukoprotease inhibitor (also called bronchial inhibitor or mucus proteinase inhibitor), which inhibits neutrophil elastase and cathepsin G, does not inhibit Pr3 (3). The 53-kD ₁PI and the 42-kD monocyte/neutrophil elastase inhibitor belong to the serpins, a superfamily of proteins with highly conserved secondary structural elements (nine -helices and three ß-sheets). The serpins form denaturant-stable complexes with their target proteinases, and behave kinetically like irreversible inhibitors. The proteinase cleaves the P₁-P^'₁ peptide bond of the inhibitor, and the serine residue of its catalytic site forms an ester bond with the P₁ residue of the inhibitor (7). The serpin ₁PI is mainly synthesized in the liver, occurs in high concentration in plasma (26 µM), and transudes from plasma into the lung epithelial lining fluid, where its concentration is still significant (4 µM [8]). It inhibits Pr3 with a second-order association rate constant k_ass of 8.1 x 10⁶ M^-1s^-1 (3). The monocyte/neutrophil elastase inhibitor inhibits Pr3 with a k_ass of 1.7 x 10⁷ M^-1s^-1 (9). It is a widely distributed intracellular protein (10) that occurs in high concentrations in neutrophils (11) but is absent in plasma (5). Ying and Simon (12) have shown that elafin is a reversible Pr3 inhibitor with a k_ass and a k_diss of 4 x 10⁶ M^-1s^-1 and 1.7 x 10^-3 s^-1, respectively. Taking into account these parameters and the in vivo concentration of inhibitor, these authors conclude that elafin plays a minor anti-Pr3 role in the lung. Thus, ₁PI appears to be the major Pr3 inhibitor in normal lung. Cigarette smoking or neutrophil-derived oxidants are able to oxidize its P₁ methionine residue (13, 14). Whereas oxidation has little effect on the inhibition of human pancreatic elastase (15), it considerably decreases the k_ass for human neutrophil elastase and cathepsin G (16). In this work we investigate whether it also affects the inhibition of Pr3.

Pr3 is an elastolytic enzyme whose intratracheal instillation in the hamster develops emphysematous lesions that resemble the human disease (2). Is the inhibition of Pr3 by ₁PI in human lung peripheral airways fast enough to prevent emphysema due to elastin degradation? We have shown previously that knowledge of the kinetic mechanism of inhibition and of the in vivo inhibitor concentration helps predict the in vivo proteolysis-preventing function of an irreversible inhibitor such as ₁PI. If the inhibition takes place in one step in vivo (E + I EI), its rate will be governed by the second-order association rate constant k_ass (17). In contrast, if it takes place in more than one step in vivo, it will be much faster than anticipated from k_ass (18). This led us to investigate the mechanism of inhibition of Pr3 by ₁PI.

	Materials and Methods

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Materials
Pr3 and ₁PI came from Athens Research and Technology (Athens, GA). Both proteins were homogeneous on native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (see below). Active site titration of ₁PI was done with titrated human neutrophil elastase as described earlier (19). Remazol-brilliant-blue-labeled bovine neck ligamentum elastin (RBB-elastin) was from Elastin Products Co. (Owenville, MO), and 4,4'-dithiopyridine andN-chlorosuccinimide from Sigma (St. Louis, MO). Methoxysuccinyl-lysine-(2-picolinoyl)-alanine-proline-valine-thiobenzyl ester (TBE) was synthesized for us by Enzyme System Products (Livermore, CA). The polyacrylamide gradient gels and the Phastsystem electrophoresis apparatus were from Pharmacia-Amersham (Uppsala, Sweden). All experiments were done in 50 mM HEPES, 150 mM NaCl, pH 7.4, a solution called "the buffer."

Elastolysis
A suspension of RBB-elastin (3 mg/ml) in the buffer was stirred at 37°C for 15 min before addition of 500 µl of buffered Pr3 ± ₁PI. From time to time, a 500-µl sample was withdrawn, added to 500 µl of 0.75 M acetate buffer pH 4.0, centrifuged at 10,000 x g for 10 min, and read at 595 nm to assay the concentration of soluble elastin peptides. Absorbance readings were done against an acidified control containing no Pr3 or ₁PI. Full solubilization of 3 mg/ml RBB-elastin yielded A_{595 nm} = 1.55, from which the percentage of elastin solubilization could be calculated.

Oxidation of ₁PI
The inhibitor was oxidized with N-chlorosuccinimide as described by Padrines and coworkers (20). The preparation was unable to inhibit porcine pancreatic elastase, but had full inhibitory activity against bovine pancreatic chymotrypsin, a characteristic feature of oxidized ₁PI (16), indicating that the N-chlorosuccinimide treatment did not denature the protein.

Substrate and Inhibitor Kinetics
The kinetic parameters k_cat and K_m describing the Pr3-catalyzed hydrolysis of TBE were determined by measuring the rates of substrate hydrolysis at 324 nm and 25°C using variable concentrations of TBE (5–200 µM) and constant concentrations of Pr3 (2 nM). The buffer contained 3 mM 4,4'-dithiopyridine, which reacts with benzyl mercaptan, the product of substrate hydrolysis, to yield 4-thiopyridone (_{324 nm} = 19,800 M^-1 cm^-1). The data were fitted to the Michaelis-Menten equation by nonlinear regression analysis (Enzfitter software; Biosoft Software, Cambridge, UK) to calculate the best estimates of the parameters and their confidence intervals.

The kinetics of inhibition of Pr3 by ₁PI was measured by adding Pr3 to a mixture of ₁PI and TBE and recording the release of product. Rapid mixing, absorbance recording, and calculation of the rate constants and their confidence intervals were done with a thermostated Bio-Logic stopped flow apparatus with a dead time of 1.7 ms and a built-in software (Bio-Logic, Claix, France).

	RESULTS

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Effect of Oxidized ₁PI on Pr3
Figure 1 shows that oxidized ₁PI has no effect on the activity of Pr3 on the synthetic substrate TBE, whereas under identical conditions, the native inhibitor readily inhibits the enzyme. Larger concentrations of oxidized ₁PI were also ineffective. Identical results were obtained with RBB-elastin as the substrate (see below). Native and oxidized ₁PI were incubated with Pr3 for variable periods of time before polyacrylamide gel electrophoresis. Figure 2 shows that the native ₁PI + Pr3 mixtures exhibited two major protein bands corresponding to unreacted 53-kD and complexed 76-kD ₁PI. In contrast, the 76-kD band was not seen in mixtures formed with oxidized ₁PI. In addition, the intensity of the 53-kD band did not change with time, indicating that the oxidized inhibitor is not a substrate of Pr3.

View larger version (13K): [in this window] [in a new window]   Figure 1. Titration of Pr3 by native (filled circles) and oxidized (open circles) 1PI. Increasing concentrations of inhibitor ([I0]) were mixed with constant concentrations of Pr3 ([E0] = 0.1 µM) at pH 7.4 and 25°C. After 1 h, 0.1 mM TBE was added to measure the residual enzyme activities. v0 and vi are the enzymatic velocities in the absence and presence of inhibitor, respectively.

View larger version (103K): [in this window] [in a new window]   Figure 2. Sodium dodecyl sulfate 8–25% polyacrylamide gel electrophoresis of mixtures of 5 µM native 1PI (A) or 5 µM oxidized 1PI (B) with 2 µM Pr3. Before electrophoresis the media were incubated at pH 7.4 and 25°C for variable periods of time. Lane 1: native or oxidized 1PI alone; lanes 2–7: Pr3 + inhibitor mixtures incubated for 5, 10, 20, 40, 60, and 180 min, respectively. The molecular weight markers were ovalbumin (45 kD) and serum albumin (66 kD). The faint bands running ahead of native 1PI + Pr3 mixtures are degradation products of the inhibitory complex also observed with other serpin–proteinase complexes. The very faint bands running ahead of oxidized 1PI are probably oxidant-denatured inhibitor.

Effect of ₁PI on the Elastolytic Activity of Pr3

View larger version (20K): [in this window] [in a new window]   Figure 3. Kinetics of solubilization of RBB-elastin by Pr3 in the absence or presence of 1PI at pH 7.4 and 37°C. Open squares, Pr3 control (0.5 µM); filled squares, 0.5 µM Pr3 + 5 µM oxidized 1PI; open circles, competition between elastin and 1PI for the binding of Pr3: RBB-elastin (3 mg/ml) and 1PI (5 µM) were stirred for 15 min before addition of Pr3 (0.5 µM); filled circles and open triangles, inhibition of elastin-bound Pr3 by 1PI: RBB-elastin (3 mg/ml) and Pr3 (0.5 µM) were stirred for 15 min before addition of 0.7 µM (filled circles) or 5 µM 1PI (open triangles).

Mechanism of Inhibition of Pr3 by ₁PI

The substrate used in the progress curve method must be sensitive enough to allow a significant amount of product to be released during the short time of the inhibition reaction, that is, it must have a high k_cat. The substrate should also have a low K_m to be used at [S]₀ > K_m, which (i) avoids significant substrate depletion during the inhibition reaction, and (ii) slows down the rate of inhibition. Such a substrate was not available at the time of the present investigation (3, 21–23). Fruh and colleagues (23) had, however, shown that methoxysuccinyl-lysine-(2-picolinoyl)-alanine-proline-valine-p.nitroanilide is a convenient substrate of Pr3 with a fairly low K_m (16 µM) but a poor k_cat (0.49 s^-1). We therefore designed the synthesis of TBE, the thiobenzyl ester derivative of their substrate. Such derivatives are known to have much higher k_cat values than p.nitroanlides (24). TBE was found to be hydrolyzed by Pr3 with k_cat = 46 ± 4.2 s^-1 and K_m = 10 ± 3 µM. This is the most sensitive chromogenic Pr3 substrate ever reported.

All progress curves were recorded using [I]₀ = 15[E]₀ and [S]₀/K_m = 5 so that no inhibitor or substrate depletion occurred during the inhibition process. Under these conditions, an irreversible inhibitor like ₁PI yields an exponential progress curve described by Equation 1 (18):

(1)

where [P] is the concentration of product at any time t, k the apparent first-order rate constant of inhibition, and v_z the rate of substrate hydrolysis at t = 0. The progress curves were recorded at 324 nm (data not shown). Their shapes were similar to those obtained for the reaction of ₁PI with other proteinases (25). They were fitted to Equation 1 by nonlinear regression analysis to calculate the best estimates of k.

Figure 4 shows that the inhibition of Pr3 by ₁PI exhibits a hyperbolic dependence of k versus [I]₀, indicating that it takes place in at least two steps, as illustrated in Scheme I:

View larger version (11K): [in this window] [in a new window]   Figure 4. Apparent first-order rate constants of inhibition of Pr3 as a function of 1PI concentration at pH 7.4 and 25°C. The rate constants k were measured as described in the text. The inhibition of Pr3 by 1PI was assessed using variable [E]0 (5–20 nM), constant [I]0/[E]0 = 15 and 50 µM TBE. Each point represents the average data collected from at least four stopped-flow traces. The line is theoretical and has been calculated using the best estimates of k2 and Ki* obtained by fitting the data to Equation 2 by nonlinear regression analysis.

(2)

where . A nonlinear fit of the data of Figure 4 to Equation 2 confirmed their adherence to the two-step model and gave the best estimates of k₂ (0.041 ± 0.003 s^-1) and K^*_i (228 ± 36 nM), a value from which K^*_i = 38 ± 6 nM was calculated. For ₁PI concentrations well below K^*_i, EI* does not accumulate significantly so that the inhibition behaves like a simple bimolecular reaction (E + I EI) governed by a second-order association rate constant .

	Discussion

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Chemical oxidation of ₁PI with N-chlorosuccinimide transforms its reactive site loop Met₃₅₁ and Met₃₅₈ residues into methionine sulphoxides and impairs its proteinase inhibitory capacity (26). Biological oxidation by the neutrophil-derived myeloperoxidase/H₂O₂ system (14), activated neutrophils (20), or environmental oxidation during cigarette smoke (13) gives similar results. Isolation of oxidized ₁PI from lung lavage fluids of smokers indicates that oxidation of the inhibitor also takes place in vivo (27). Oxidized ₁PI reacts much slower, but still forms tight complexes with neutrophil elastase and cathepsin G (16, 20). In contrast, it does not react with neutrophil Pr3, as shown in this paper. This observation has important bearing on the pathogenesis of destructive lung diseases. Pr3 cleaves a number of extracellular matrix proteins (3) and induces lung emphysema in the animal (2). Unlike neutrophil elastase, it is not inhibited by the secretory leukoprotease inhibitor present in airways secretions (3). Therefore, if part of lung ₁PI is oxidized in the vicinity of activated neutrophils, Pr3 may attack lung elastin and other extracellular matrix proteins in an unimpaired way. This enzyme may, therefore, play a prominent tissue-destructive role in lung emphysema and cystic fibrosis. There is, however, only circumstantial evidence for the role of Pr3 in these diseases (2).

If Pr3 is released in the vicinity of elastin fibers and in the presence of active ₁PI, it theoretically partitions between its substrate and its inhibitor. It is likely that this partition is largely in favor of ₁PI in vivo because our in vitro experiments show that there is no elastolysis if Pr3 is added to a mixture of elastin and ₁PI. On the other hand, neutrophil elastase is bound to lung connective tissue in close proximity to elastic fibers following intratracheal instillation in the animal (28). A similar location in the human emphysematous lung (29) has been controverted (30). It stands, however, to reason that involvement of neutrophil proteinases in emphysema assumes binding of these enzymes to elastin fibers. This raises the question of whether ₁PI is able to inhibit elastin-bound elastase and Pr3. In a previous paper we have shown that elastin-bound elastase was not fully inhibited by ₁PI even after 150 min of reaction (31). With Pr3 we arrive at a similar but not identical conclusion: in the presence of elastin, ₁PI becomes a slow-binding Pr3 inhibitor that requires a certain time to fully inhibit the enzyme. We also show that the amount of elastin solubilized during this lag time depends on the concentration of ₁PI. While this work was in progress, Ying and Simon (32) stated that elastin-bound Pr3 is fully inhibited by ₁PI. They used an end-point elastolytic assay, which is less reliable than our kinetic assay. They also noticed that a 1.5-molar excess of inhibitor over enzyme was required to observe full inhibition of elastolysis, indicating that their results do not fully conflict with ours.

Rao and coworkers (3) were the first to demonstrate the inhibition of Pr3 by ₁PI and found k_ass = 8.1 x 10⁶ M^-1s^-1, a value 8-fold higher than our k_ass calculated from k₂/K^*_i. It is likely that this discrepancy rests on methodological errors. Rao and colleagues (3) used a discontinuous method in which equimolar concentrations (8.5 nM) of Pr3 and ₁PI were incubated for variable periods of time before addition of substrate, which measures the residual enzymatic activities and should stop the association process. We have previously emphasized that two requirements should be met to get reliable k_ass values using this discontinuous method: (i) t_1/2, the half-life of the association (t_1/2 = 1/k_ass[E]₀) should be larger than 2 min to minimize sampling errors, and (ii) the substrate should be used at a concentration well above K_m to efficiently stop the association. None of these requirements were met in the method described by Rao and coworkers (3). Also, these authors did not measure the rate of inhibition as a function of ₁PI concentration and could, therefore, not detect the reaction intermediate EI* evidenced in the present article.

Is ₁PI able to efficiently protect lung extra cellular matrix proteins against proteolytic degradation due to Pr3? Our kinetic data may help answer this question. We have shown that the formation of the final irreversible EI complex is preceded by a fast building up of a tight-binding reversible inhibitory EI* complex. Because the ₁PI concentration of lung epithelial lining fluid (4 µM [8]) is 120-fold higher than K^*_i, it may be calculated (18) that the initial E + I EI* equilibrium is almost fully shifted toward EI*. Thus, immediately after its release from neutrophils, Pr3 will be almost fully taken up as an EI* complex. This will greatly reduce its binding possibilities to extracellular matrix proteins or to neutrophil membrane (4). Neutrophil elastase and cathepsin G do not share this property. Elastase does not form an EI* complex in vitro, even with inhibitor concentrations as high as 6 µM (33). Although this complex forms with cathepsin G, its K^*_i (0.8 µM [25]) is too high to allow formation of significant amounts of EI* in the epithelial lining fluid.

The proteolysis-preventing function of a proteinase inhibitor also depends on the rate constant of proteinase inhibition in vivo (17). For two-step inhibition, this corresponds to k*, the rate constant of EI* formation: k* = k₁[I]₀ + k_-1 which may also be written as . Because accumulation of EI* requires k_-1>> k₂, one gets . Using [I]₀ = 4.6 µM, k₂ = 0.041 s^-1, and . Thus, d(t), the delay time of inhibition (17) of Pr3 by ₁PI in the epithelial lining fluid (d(t) = 7 t_1/2), is much lower than 0.94 s, and is perhaps in the millisecond range. If Pr3 and ₁PI would react in vivo with the same k_ass but without forming an EI* complex, the delay time of inhibition would be 1.1 s. This illustrates the outstanding physiological advantage of multi-step inhibition (18).

	Acknowledgments

 
The authors thank the association "Vaincre la Mucoviscidose" for financial support.

Received in original form November 19, 2002

Received in final form January 9, 2003

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