Biol. Chem. Hoppe-Seyler Vol. 372, pp. 13-21, January 1991

Purification and Characterization of Elastase-Specific Inhibitor Sequence Homology with Mucus Proteinase Inhibitor Jean-Michel SALLENAVE and Andrew P. RYLE Department of Biochemistry, Edinburgh University Medical School

(Received 4 September 1990)

Summary: Elastase-specific inhibitor (ESI) was purified from sputum of patients with chronic bronchitis and compared with mucus proteinase inhibitor (MPI, BrI) isolated, without the use of affinity chromatography on an enzyme, from non-purulent sputum of a patient with bronchial carcinoma. The N-terminal sequence of 27 residues of the latter was determined and showed serine as the only N-terminus.The partial

N-terminal amino-acid sequence of ESI shows some homology with MPI, especially around the reactive site of MPI for human neutrophil elastase.This region could therefore be the reactive site of ESI. The thermodynamic and kinetic constants of the reactions of ESI with human neutrophil elastase and with porcine pancreatic elastase show that ESI is a fast-acting inhibitor.

Reinigung und Charakterisierung eines Elastase-sp.ezifischen Inhibitors und seine Sequenzhomologie mit dem Mucus-Proteinase-Inhibitor Zusammenfassung: Elastase-spezifischer Inhibitor (ESI) wurde aus dem Sputum von Patienten mit chronischer Bronchitis isoliert und mit dem Mucus-Proteinase-Inhibitor (MPI, BrI) aus dem nicht eitrigen Sputum eines Patienten mit Bronchialkarzinom verglichen, der ohne Affinitätschromatographie an einem Enzym isoliert wurde. Die N-terminale Sequenz von MPI wurde über 27 Reste bestimmt und zeigte Serin als einzigen N-Terminus. Die partielle N-termi-

nale Aminosäuresequenz von ESI zeigt einige Homologien mit MPI, besonders im Bereich von dessen reaktivem Zentrum für neutrophile Elastase. Von daher erscheint ein Rückschluß auf das reaktive Zentrum von ESI möglich. Die thermodynamischen und kinetischen Konstanten der Reaktionen von ESI mit menschlicher neutrophiler Elastase und mit der Pankreas-Elastase vom Schwein zeigen, daß ESI ein schnell wirkender Inhibitor ist.

Key terms: Mucus proteinase inhibitor, elastase-specific inhibitor, sequence, kinetics, sputum.

Enzymes: Cathepsin G (EC 3.4.21.20); Chymotrypsin (EC 3.4.21.1); Elastase (porcine pancreatic) (EC 3.4.21.36); Elastase (human neutrophil) (EC 3.4.21.37); Trypsin (EC 3.4.21.4). Abbreviations: MPI: mucus proteinase inhibitor; ESI: elastase-specific inhibitor; BrI: bronchial inhibitor; arPI: alphai-proteinase inhibitor; HNE: human neutrophil elastase; PPE: porcine pancreatic elastase; Bz-L-Arg-OEt: benzoyl-L-arginine ethyl ester; S-2484: L-pyroglutamyl-L-prolyl-L-valyl-4-nitroanilide; Slapn: succinyl tri-L-alanine-4-nitroanilide; ELISA: enzyme-linked immunosorbent assay; PAGE: polyacrylamide-gel electrophoresis; SDS: sodium dodecyl sulphate;TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; FPLC: fast protein liquid chromatography (Pharmacia).

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14

J.-M. Sallenave and A.P. Ryle

Human bronchial mucous secretions have been shown to contain inhibitors of serine proteinases secreted by neutrophils; their role is probably to control the enzymes both in the upper airways and in the lung interstitium. Three of these inhibitors have been identified and characterized to varying degrees: o^-proteinase inhibitor, mucus proteinase inhibitor (also called bronchial inhibitor or antileukoprotease), and elastasespecific inhibitor. There is conflict in the literature over the presence and importance of MPI in the peripheral airways: compare refsJ1"^. To date, «i-PI seems to be the major serine proteinase inhibitor present in the peripheral airways (MPI/arPI molar ratio ranging from 0.02 to 0.14), whereas in the large airways, a ratio around 1.4 is found[4]. MPI has been shown to be secreted by the serous cells of submucosal glands of both the upper and lower airways151 and by Clara cells in the bronchiolar epithelium161. Nevertheless, the composition of lavage fluid does not necessarily reflect that in the interstitium and although it is present in alveolar lavage in minute amounts, compared to arPl. MPI might still have a protective role in the interstitium: Willems et al.[?1 have shown that it is associated with elastic fibres in the alveolar septum, presumably for a protective role. Another possibly important proteinase inhibitor, although less well known, is the elastase-specific inhibitor, first described in 1981 by Hochstrasser et al.[8] and then is 1985 by Kramps and Klasen[yJ. Although present in lower concentrations than MPI in bronchial secretions (molar ratio = 0.1^), it is more specific for elastolytic enzymes as it inhibits human neutrophil elastase and porcine pancreatic elastase but not trypsin, chymotrypsin or cathepsin G[8'9]. Although no sequence was available, Hochstrasser et al. noted some similarity in the amino-acid composition of their elastase-specific inhibitor (called BSI-E) compared to their BSI-TE (now recognised[10] to be identical to MPI). It was the aim of this work to purify both ESI and MPI from sputum and to compare their sequences and their biochemical properties. A preliminary account of this work has appeared^111.

Materials and Methods MATERIALS Rabbit anti-MPI IgG was kindly given by Dr. H.J. Kramps, Leiden, The Netherlands; sheep anti-(rabbit y-globulin) conjugated to horse-radish peroxidase was obtained from Seralab, Crawley Down, Sussex RH10 4FF.The substrate S-2484 was supplied by Kabi-Vitrum, Stockholm, Sweden, Slapn by Peptide Institute Inc, Osaka, Japan. Bz-Arg-OEt, myoglobin fragments as molecular mass standards and bovine serum albumin came from Sigma, Poole, Dorset.

Vol. 372 (1991)

METHODS Estimation of protein Protein concentrations were measured by the method of Bradford'121 with bovine serum albumin as standard: on chromatograms the absorbance at 280 nm was used. Determination ofamino acid sequences Sequences were determined for us by the Welmet Protein Characterisation Facility housed within the Department. Polypeptides were routinely checked for purity by reverse-phase chromatography on an Aquapore RP-300 column (Applied Biosystems: 7 μπι particle size; 2.1 mm x 30 mm). The column was developed at 0.2 m//min in 0.1% (v/v)TFAby a linear 8-80% (v/v) acetonitrile gradient over 50 min. The eluate was monitored continuously at 220 nm. The pure polypeptides were subjected to automated sequencing on an Applied Biosystems 477A instrument, after reduction and pyridylethylation of the cysteine residues'131. Determination of reactive site concentrations The reactive site concentration of bovine pancreatic trypsin (Sigma) was determined by reactive site titration by the method of Chase and Shaw'141 to be 60% and it was used to titrate a stock solution of a r PI (Sigma) which in its turn was used as a secondary standard for the reactive site titration of HNE and PPE. Various amounts of a r PI were incubated for 30 min with 0.09 nmol of active bovine trypsin in 10mM Tris/HCl, pH 8.0, in a total volume of 133 μ/. The residual enzyme activity was measured by adding 0.867 ml of Bz-L-Arg-O-Et (Sigma) to the reaction mixture (final concentration: 0.46mM) and recording the increase in absorbance at 253 nm. arPI was determined by this method to be 53% active. Similar experiments were performed with α Γ ΡΙ to determine the reactive site concentrations of HNE and PPE. Inhibitor and enzyme mixtures were incubated for a period of 30 min.*The HNE used in our experiments, purified according to Martodam et al.'15' was found to be 50% active.The PPE (Sigma) was found to be 65% active. Detection of inhibitors During the purification of ESI and MPI, PPE and HNE respectively were used to detect the active inhibitors, though in order to conserve material quantitative assays were not done. The putative inhibitor samples were reacted for 30 min with constant amounts of HNE (to give about 10nM initial active enzyme in the final incubation with substrate) in 110 μΐ of 0.05 Μ Tris/HCl, 0.5M NaCl, pH 8.0 (buffer 1) at 37 °C. The remaining enzymic activity was measured by addition of 56 μΐ of 8mM-L-pyroglutamyl-L-prolyl-L-valyl-4-nitroanilide (S-2484) and 945 μι of buffer 1 with recording at 405 nm. To detect PPE-inhibitory activity, the same procedure was followed except that the initial active concentration of PPE was about 20nM. The remaining activity was measured by addition of 56 μΐ of 20mw succinyl-L-Ala-L-Ala-L-Ala-4-nitroanilide (Slapn). Determination of molecular mass This was done by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate and urea by the method of Swank and Munkres'161 as modified in Sigma bulletin No. MWS-877P and molecular mass assignments were made by using plots of mobility against log m obtained for standard polypeptides, i.e. myoglobin

* There are no signs of degradation of «i-PI. The long period time used was to ensure complete inhibition.

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Vol. 372 (1991)

15

Elastase-Specific Inhibitor

fragments (2510; 6210; 8160; 14400; 16950 Da), cytochrome c (12400 Da), lysozyme (14300 Da). The bands were detected by silver staining[17].

is inappropriate for the same reasons as for use of the Easson-Stedman equation for determination of K\).

Electrophoretic transfer onto nitrocellulose sheets

Results

Western-blot analysis was performed by the method of Towbin et al.[18] and antigenic bands were detected with a 1/500 dilution of a rabbit anti-MPI IgG, followed by 1/500 dilution of a sheep anti-rabbit IgG antibody conjugated to horseradish peroxidase. Dilutions were made in 20mM Tris/HCl, 0.15M NaCl, 0.1% Tween 20, pH 7.4. The Western blots were developed by adding the freshly-prepared substrate (0.06% (w/v) 4-chloro-l-naphthol, 0.01% H2O2) in a solution of 50mMTris/HCl, 150mM NaCl, 20% methanol, pH 7.4.

Isolation and purification of elastase-specific inhibitor (ESI) ESI was purified from sputum which was collected daily from patients with chronic bronchitis and stored at - 20 °C.The method used was a modification of the one described by Kramps and Klasen^ which includes affinity chromatography on immobilised PPE in order to remove MPI. However, the exclusion chromatography on Sephadex G-75 was omitted, the cation-exchange chromatography was done on SESephadex C-50 (rather than on SP-Sephadex C-50) and final purification was done by reverse-phase chromatography.

Determination of the dissociation constant, K,, and titration of inhibitor Reaction mixtures were made up with varying amounts of purified inhibitor as for detection of inhibitor (above) except that reactive site-titrated HNE or PPE were used to give initial active concentrations in the final assay mixture of 9.7nM and 20.5nM respectively. Residual enzyme activity was measured as above and used to plot the inhibition curve and hence determine the molecular mass of the inhibitor. Although it is common practice to obtain the value of K\ from the slope of a straight line fitted by linear regression of the EassonStedman equation'19', the practice is not justified since it is most unlikely that the values of I/a (where a is the fractional activity remaining) are normally distributed; furthermore the dependent variable, a, appears on both sides of the equation. We have therefore fitted the solution to the equation e~ - (e() - K\ - i) x e - e0 x K{ = 0 e = ((