©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Influence of Low Molecular Mass Heparin on the Kinetics of Neutrophil Elastase Inhibition by Mucus Proteinase Inhibitor (*)

Martine Cadène (1), Christian Boudier (1), Guy Daney de Marcillac (2), Joseph G. Bieth (1)(§)

From the (1) Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, F-67400 Illkirch, France and the (2) Institut de Biologie Moléculaire des Plantes, CNRS, F-67000 Strasbourg, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Commercial low molecular mass heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor, the predominant antielastase of lung secretions (Faller, B., Mély, Y., Gérard, D., and Bieth, J. G.(1992) Biochemistry 31, 8285-8290). To study the kinetic mechanism of this rate enhancement, we have isolated a 4.5-kDa heparin fragment from commercial heparin. This compound is fairly monodisperse as shown by analytical ultracentrifugation. It binds elastase and inhibitor with a 1:1 stoichiometry and an equilibrium dissociation constant of 3 and 210 nM, respectively. It also forms a tight complex with EI. Flow calorimetry shows that the inhibitor-heparin interaction is characterized by a large negative enthalpy change (H = -45.2 kJ mol) and a small entropy change (S = -23.7 J K mol). Stopped-flow kinetics run under pseudo-first-order conditions ([I] [E]) show that in the absence of heparin the inhibition conforms to a simple bimolecular reaction,

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

where, k = 3.1 10Ms, k= 10 s, and K= 33 pM, whereas in the presence of heparin, E and I react via a two-step mechanism,

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

where K * = 86 nM, k = 2.2 s, k = 10 s, and K= 37 pM. Thus, heparin increases both the rate of inhibition by promoting the formation of a high affinity EI* intermediate and the rate of EI dissociation. Since the dissociation is negligible in bronchial secretions where the inhibitor concentration is much higher than K, it may be concluded that heparin significantly potentiates the inhibitor's antielastase potential in vivo.


INTRODUCTION

Neutrophil elastase (NE)() is an essential component of the phagocytic machinery of polymorphonuclear leukocytes. This 30-kDa serine proteinase is a cationic glycoprotein formed of 218 amino acid residues with 19 arginines but no lysines (1) . X-ray crystallographic studies show that the arginine residues are all located in patches at the surface of the protein (2, 3) . NE has a number of physiologic functions including proteolysis of phagocytosed proteins, neutrophil migration, and tissue remodelling following injury (4). It also cleaves extracellular matrix proteins such as elastin, interstitial and type IV collagens, proteoglycans, laminin, and fibronectin as well as plasma proteins such as antithrombin, immunoglobulins, fibrinogen, and components of the complement system (5).

The concentration of NE in the azurophil granules of polymorphonuclear leukocytes is thought to be in the millimolar range (6) . An efficient anti-NE control system must therefore be present at inflammatory sites where neutrophils are recruited and activated or where they die in order to prevent undesirable degradation of the aforementioned proteins. Healthy individuals have efficient NE inhibitors including -proteinase inhibitor, -macroglobulin, mucus proteinase inhibitor (MPI), (5) and elafin (7) . The two former proteins are present in plasma and are able to diffuse in the interstitial fluid while the latter are found in airway or genital tract secretions and in skin (for review see Refs. 5 and 7).

The 53-kDa glycoprotein -proteinase inhibitor is the most important NE inhibitor on a molar basis. In addition, it inhibits NE irreversibly with a very high association rate constant, k> 10M s(8, 9) . It therefore acts in vivo as an extremely fast and efficient NE inhibitor. Its biological NE inhibitory function is best illustrated by the fact that its hereditary deficiency frequently leads to pulmonary emphysema, a disease characterized by lung elastin and collagen fiber disruption with subsequent airflow limitation.

MPI is the most abundant physiologic NE inhibitor of the upper respiratory tract, where it occurs in concentrations as high as 5 µM(10, 11) . It is an 11.7-kDa basic protein whose structure is stabilized by eight disulfide bonds. It is formed of a single chain of 107 amino acid residues of known sequence (12, 13) and is composed of two domains of similar size and architecture (14) . The x-ray diffraction study on the MPI-chymotrypsin complex has identified Leu as the P1 residue of the inhibitor's active site. NE binds at the same site as chymotrypsin (15) .

The inhibition of NE by both -proteinase inhibitor and MPI can be modulated by heparin, an anionic glycosaminoglycan synthesized by mast cells. This polymer mostly consists of trisulfated disaccharide units formed of L-iduronic acid-2-sulfate linked to D-glucosamine-N,3,6-sulfate (16) . It modulates the activity of numerous biological systems (17) . Among others, it binds and activates a number of protein proteinase inhibitors that belong to the serpin superfamily, namely antithrombin, heparin cofactor, protease nexin, protein C, and plasminogen activator inhibitor (17, 18) . Heparin does not bind -proteinase inhibitor, another serpin, but strongly decreases the rate of NE inhibition by this inhibitor as a result of its strong binding with the enzyme. In contrast, the polymer strongly binds MPI, a non-serpin inhibitor and significantly increases its rate of reaction with NE. It also increases the intrinsic fluorescence of the inhibitor, blue-shifts its maximum emission wavelength, and increases its fluorescence lifetime, suggesting that it buries Trp, the single tryptophan residue of the protein in a very hydrophobic environment (19).

In our previous work, we used a heparin preparation qualified as ``5.1-kDa heparin'' by the manufacturer. Such a material, known as low molecular mass heparin, is obtained by controlled chemical depolymerization of standard heparin and is erroneously believed to be less polydisperse than standard heparin. We therefore decided to use the 5.1-kDa heparin to purify a polymer with a low degree of dispersity. This material was then used to assess the kinetic mechanism of the activation of MPI by heparin.


EXPERIMENTAL PROCEDURES

The source of NE, MPI, and commercial 5.1-kDa heparin was the same as before (19) . Suc-Ala-pNA and MeO-Suc-Ala-Pro-Val-pNA were from Bachem, Switzerland, while MeO-Suc-Ala-Pro-Ala-thiobenzylester came from Enzyme System Products (Livermore, CA). Stock solutions of most substrates and of 4,4`-dithiodipyridine (Sigma) were made in dimethylformamide, while Suc-Ala-pNA was dissolved in N-methylpyrrolidone. The final concentration of organic solvent was 2% (v/v) throughout. Unless otherwise stated, all experiments were performed at 25 °C in 50 mM Hepes, 100 mM NaCl, pH 7.4, a solution that will be referred to as the ``buffer.'' The buffer was filtered through a 0.45-µm microporous membrane and degassed prior to use. Except for the stopped-flow experiments, all regression analyses were done using the Enzfitter software.

Purification of Heparin

One gram of commercial 5.1-kDa heparin was chromatographed on a 5 190-cm column of Sephadex G-50 Superfine (Pharmacia Biotech Inc.) eluted with a 200 mM NaCl solution at a flow rate of 22 ml/h. The collected fractions (11 ml) were tested for the presence of heparin using the azure A blue (Sigma) assay (20) . Heparin eluted as a broad peak, which was divided into seven portions, each corresponding to an elution volume of about 80 ml. These fractions were lyophilized, dissolved in a minimal volume of deionized water, and desalted on a Sephadex G-10 column. The salt-free fractions were concentrated by lyophilization and rechromatographed on Sephadex G-50 as described above. The tubes containing the highest concentrations of heparin were pooled, desalted, and lyophilized. The material issued from the last fraction of the initial chromatography is referred to as ``heparin'' in the text and was used throughout the present investigation.

Ultracentrifugation

Ultracentrifugation of heparin was performed at 20 °C using a Beckman Model E ultracentrifuge equipped with a Schlieren optical system and a camera. One hundred forty µl of a 1 mg/ml heparin solution in 200 mM NaCl were injected in one compartment of a double sector cell while 160 µl of 200 mM NaCl were injected in the other compartment. The ultracentrifuge was run at 5,000 rpm (diffusion coefficient) or 50,000 rpm (sedimentation coefficient). A number of photographs of the Schlieren patterns were taken at regular intervals of time over a period of 35 min. The sedimentation and diffusion coefficients were determined by graphical methods based on classical equations. The molecular mass was calculated using Svedberg's equation, a partial specific volume of 0.42 ml g(21) , and a solution density of 1.01.

High Performance Liquid Chromatography

The molecular mass of heparin was also determined by chromatography on a 60 0.75-cm TSK 3000 SW column (Touzart et Matignon, France) eluted with 200 mM NaCl at a flow rate of 0.5 ml/min. The eluted heparin was detected using a differential refractometer from Knauer (Berlin). The column was calibrated with 2.6-9.5-kDa heparin standards (a gift from Dr. Petitou, Sanofi, France) whose molecular masses had been determined by ultracentrifugation (22) .

The Heparin-Protein Binding Stoichiometry

The heparin-protein binding stoichiometry was determined using the ability of a heparin-binding dye to displace heparin from its complex with a heparin-binding protein (23) . The dye was azure A blue (20) instead of proflavine (23) because the latter has a poor color yield with our heparin preparation. With 150 µM azure A blue the absorbance at 595 nm decreased linearly when the heparin concentration increased from 0.07 µM to 3 µM, thus making the stoichiometry determination possible. Constant concentrations of heparin (2 µM) were preincubated for 5 min at room temperature with variable concentrations of NE, MPI, or NEMPI complex in the buffer. Fifty µl of these mixtures were then added to a cuvette containing 950 µl of a solution of 150 µM azure A blue dissolved in the buffer. Absorbances were measured at 595 nm against a 150 µM azure A blue control solution.

Flow Calorimetry

Flow calorimetry was used to measure the K of the MPI-heparin complex. Equal volumes of MPI and heparin buffered solutions were injected at a flow rate of 0.198 ml min into a ThermoMetric 2277 Thermal Activity Monitor. The power was recorded until the thermal equilibrium was reached (about 20 min). The equilibrium power p was measured at 25 °C using constant concentrations of MPI (2 µM) and variable concentrations of heparin (0.5-10 µM). The data were analyzed by nonlinear regression based on the equation,

On-line formulae not verified for accuracy

where p is the extrapolated power at infinite heparin concentration, and [I] and [H] are the total MPI and heparin concentrations, respectively.

Enzymatic Methods

The kinetic constants k and K were measured for the various substrates of NE in the presence and absence of heparin as described previously (24) .

The effect of heparin on the stoichiometry and the equilibrium dissociation constant K of the NEMPI complex was determined by reacting constant concentrations of NE with increasing concentrations of MPI in the absence or presence of a constant concentration of heparin for a given time and measuring the residual enzymatic activity with a synthetic substrate. The stoichiometry was determined using 0.2 µM NE with or without 40 µM heparin, an incubation time of 5 min at 25 °C, and 1 mM Suc-Ala-pNA (25) . The K was measured with 20 nM NE with or without 40 µM heparin, an incubation time of 15 min at 25 °C, and 4 mM MeO-Suc-Ala-Pro-Val-pNA (26) .

The rate of inhibition of NE by MPI was measured using the progress curve method (27, 28) . NE with or without heparin was added to a buffered solution of MPI, 0.3 mM MeO-Suc-Ala-Pro-Ala-thiobenzylester (29) , and 3 mM 4,4`-dithiodipyridine (30) . The reactions were followed using the same stopped-flow apparatus as previously (9) .


RESULTS

Molecular and Protein-binding Properties of Heparin

Fig. 1 shows that heparin yields symmetrical and gaussian Schlieren patterns during low speed ultracentrifugation, indicating that it is fairly monodisperse. Its M was 4500 ± 500 and 5200 ± 800 as determined by ultracentrifugation and high performance liquid chromatography, respectively. Heparin bound free NE and MPI with a 1:1 stoichiometry and the NEMPI complex with a 2:1 stoichiometry (Fig. 2). The K of the MPI-heparin complex, determined by microcalorimetry, was found to be 210 ± 30 nM, which corresponds to G = -38.2 kJ mol. On the other hand, H = -45.2 kJ mol as derived from p (Equation 3), this leading to S = -23.7 J K mol. The K of the NE-heparin complex, determined as described previously (24) , was found to be 3 ± 0.4 nM. A published program for computing distribution diagrams for multiple equilibria (31) was used to calculate the degree of saturation of NE and MPI by heparin in mixtures of the three partners. It was found that for any experimental condition described in this paper, 40 µM heparin saturates both NE and MPI to at least 99%.


Figure 1: Schlieren patterns photographed during the determination of the diffusion coefficient of heparin using low speed ultracentrifugation.




Figure 2: Stoichiometry of binding of heparin to NE (), MPI (), and the NEMPI complex (). Increasing concentrations of proteins were mixed with a constant concentration of heparin (2 µM), and the mixtures were reacted with azure A blue and read at 595 nm against a dye control. Relativeabsorbance, absorbance of the heparin + protein + azure A blue/absorbance of the free heparin + azure A blue.



Effect of Heparin on the Stoichiometry and the K of NEMPI Complex

MPI is a reversible competitive inhibitor of NE (32). Use was made of this property to infer the stoichiometry and the K of the NEMPI complex from enzyme:inhibitor titration curves (33) . The binding stoichiometry was determined using a relatively high enzyme concentration (0.2 µM) to favor the enzyme-inhibitor association and a relatively low substrate concentration ([Suc-Ala-pNA] = 1 mM, K= 0.55 mM and 3.6 mM in the absence and presence of heparin, respectively) to avoid excessive complex dissociation. Under these conditions straight inhibition curves were obtained in the absence and presence of heparin (see Fig. 3, insets). These curves indicate that the NE:MPI binding stoichiometry is 1:1 whether heparin is present or not. The K was determined under conditions that disfavored enzyme-inhibitor binding ([NE] = 20 nM) but favored complex dissociation, i.e. [S]/K 1 ([MeO-Suc-Ala-Pro-Val-pNA] = 4 mM, K= 0.085 mM and 0.123 mM in the absence and presence of heparin, respectively). Now concave titration curves were obtained (Fig. 3). The inhibition data were fit to Equation 4 (33) by nonlinear regression analysis.

On-line formulae not verified for accuracy

where a = rate in the presence of MPI/rate in its absence and K = K (1 + [S]/K ). The concave inhibition curves shown in Fig. 3are theoretical and have been calculated using Equation 4 and the best estimates of K (app). There is a good fit between these curves and the experimental points. The K values were 33 ± 9 pM in the absence of heparin and 37 ± 7 pM in the presence of heparin. The polymer, therefore, does not significantly alter the enzyme-inhibitor affinity.


Figure 3: Effect of heparin on the stoichiometry () and the equilibrium dissociation constant K of NEMPI complex () at pH 7.4 and 25 °C. The experiments were done as described under ``Experimental Procedures.'' Relativeactivity, rate in the presence of MPI/rate in its absence.



Effect of Heparin on the Rate of Inhibition of NE by MPI

Fig. 4 shows typical progress curves recorded upon addition of NE to a mixture of MPI and substrate in the absence of heparin and in the presence of a saturating concentration of the polymer. These curves were analyzed by nonlinear regression (19) to calculate k, the apparent first-order rate constant of inhibition. This constant was found to be 0.05 and 0.40 s in the absence and presence of heparin, respectively.


Figure 4: Progress curves for the inhibition of 30 nM NE by 300 nM MPI at pH 7.4 and 25 °C in the absence or presence of 40 µM heparin.



The reversible competitive inhibition process may be described by Fig. SIor II (34) , where E stands for NE or the NE-hep