178

Biochimica et Biophysica Acta, 1038 (1990) 178-185 Elsevier

BBAPRO 33620

Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain * Johannes A. Kramps 1, Charlotte van Twisk 1, Heribert Appelhans 2, Barbara Meckelein 2, Theo Nikiforov 2 and Joop H. Dijkman 1 1 Department of Pulmonology, University Hospital Leiden, Leiden (The Netherlands) and Instztut fur Btochemie, Technische Hochschule Darmstadt, Darmstadt (F.R.G.) 2

.

(Received 15 September 1989)

Key words: Antileukoprotease; Proteinase inhibitor; Methionine

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg2°-Tyr2t in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met 73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H20 2 and CI-, or c/s-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAblS) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.

Introduction

Antileukoprotease (ALP), also called secretory leukocyte proteinase inhibitor (SLPI), is produced by several types of secretory cells of the human body [1-3] and can be found in exocrine fluids including nasal and bronchial secretions, saliva, tears, cervical mucus and seminal plasma [1,4]. It is a serine proteinase inhibitor, showing strong affinity for trypsin, chymotrypsin and the neutrophil proteinases elastase and cathepsin G [5,6]. Most likely, the physiological function of ALP is to protect tissues against lysosomal proteinases released

Abbreviations: ALP, antileukoprotease; SLPI, secretory leukocyte proteinase inhibitor; PBS, phosphate-buffered saline; MoAb, mono-

clonal antibody. Correspondence: J.A. Kramps, Department of Pulmonology (C3-P), University Hospital Leiden, Rijnsburgerweg 10, 2333 Ant Leiden, The Netherlands. * Part of this work has been presented at the American Thoracic Society Meeting in Cincinnati (14-17 May 1989).

by the neutrophils during inflammatory processes. The ALP molecule, a monomer with a length of 107 amino acid residues (molecular mass 11726 Da), comprises two domains of equal length, which are homologous to each other and which are thought to be separate functional units [7,8]. Stetler et al. [9] reported that the organization of the exons and introns in the ALP gene precisely reflects the domain structure of the protein. Based on limited homology with known proteinase inhibitors, it is assumed that the antitrypsin activity is located in the first NH2-terminal domain (Leu19-Arg 2°Tyr21), whereas the anti-elastase and anti-chymotrypsin activities are located in the COOH-terminal domain of the ALP molecule (Leu72-Met73-Leu TM) [7,8]. However, in a previous study performed with ALP from human seminal plasma (HUSI-I), there was no indication that an arginine residue is involved in the activity against trypsin [10]. Recent results, obtained by X-ray crystallographic techniques, have shown that ALP consists of two well-separated domains of similar architecture, and that the chymotrypsin binding property is indeed located in the second domain [11]. Moreover, Stetler et al. [12]

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

179 and Appelhans et al. [13] were able to express the DNA sequence encoding the second domain of ALP, resulting in a protein active against neutrophil elastase. In the present study we obtained evidence, in contrast to what is generally thought, that inhibitory activities against chymotrypsin, neutrophil elastase and trypsin are all located in the second domain of the native ALP molecule. Materials and Methods

Human neutrophil elastase was isolated from purulent sputum [14,15], and titrated using the active-site titrant N-benzyloxycarbonyl-alanyl-alanyl-prolyl-azaalanine-p-nitrophenylester (Z(Ala) 2ProazaAla-ONp: Enzyme Systems Products, Livermore, CA) as described by Powers et al. [16]. Bovine pancreatic trypsin (type III), bovine pancreatic a-chymotrypsin (type II), catalase (type C-40), succinyl-alanyl-alanyl-prolyl-phenylalanine-para-nitroanilide (Suc(Ala)2ProPhe-pNA), 5bromo-4-chloro-3-indolyl phosphate and Nitro blue tetrazolium were obtained from Sigma Chemical Co., St. Louis, MO. N-a-Benzoyl-L-arg)nine-p-nitroanilide ( Na-benzoyl-Arg-pNA) was from Merck, Darmstadt, F.R.G.; benzoyMsoleucyl-glutamyl-glycyl-arginine-pnitroanilide (benzoyl-Ile-Glu-Gly-Arg-pNA) from Protogen, Laeufelfingen, CH and pyro-glutamyl-prolylvaline-p-nitroanilide (pyro-GluProVal-pNA; $2484) from Kabi Vitrum Diagnostica, Stockholm, Sweden. cis-Platinum(II)diammine dichloride ( cis-platinum) was purchased from Bristol-Myers Co., Syracuse, NY.

Isolation of human ALP from bronchial secretions ALP was isolated from perchloric acid-treated nonpurulent sputum by an ALP-specific immunoadsorbent as described previously [17,18]. Dodecyl sulphate electrophoretic analysis of purified ALP revealed one band showing an apparent molecular mass of 14 kDa.

Recombinant second-domain of ALP Expression of the second domain in Escherichia coli was performed by using the gene fragment encoding the amino acids 49-107 of ALP, as obtained by BamHIBc/I restriction of the ALP cDNA [19,20].

Displacement of trypsin from native ALP by neutrophil elastase Bovine pancreatic trypsin (0.1 nmol active enzyme) was mixed with 0.36 nmol active ALP in 240/~1 buffer (0.5 M Tris/HC1 (pH 7.8) containing 0.1% gelatin). After an incubation of 10 min at 37 ° C, 200/~1 human neutrophil elastase in saline containing 0.1% gelatin were added. Absolute amounts of elastase ranged from 0-1.70 nmol active enzyme. After another 10 min at 37 o C, the trypsin activity in the trypsin/ALP/elastase mixture was measured by adding 650 /~1 N-a-benzoyl-

Arg-pNA (2 mM in H20) to 340/~1 of the mixtures [21]. The increase in absorbance was then measured at 405 nm. Control experiments were performed by omitting either ALP or both trypsin and ALP from the mixtures. The latter experiment was performed to verify the absence of hydrolysis of trypsin substrate by elastase. The remaining 100/xl of the mixtures were used to measure the elastase activity as described previously using pyroGluProVal-pNA as substrate [22]. The capacity of an ALP-elastase complex to inhibit trypsin was measured using the same inhibition assay as described above. The ALP-elastase complex was prepared by adding 0.3 nmol ALP to 0.45 nmol neutrophil elastase. After 10 rain at 37°C, 0.15 nmol trypsin was added. This mixture was incubated for 10 rain at 37 ° C, after which time the trypsin activity was measured. A control experiment was performed by measuring the trypsin inhibition activity of free ALP.

Effect of human neutrophil myeloperoxidase on ALP inhibitory activity ALP (3.3 #M), dissolved in phosphate-buffered saline (PBS) of pH 6.0, was incubated with 213/~M H202 and human neutrophil myeloperoxidase (kindly provided by Dr. D. Roos; Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, The Netherlands). Myeloperoxidase concentrations ranged from 1-10 nM. After 8 min at 37 o C, 0.2 mg/ml catalase and methionine were added to a final concentration of 5 mM in order to stop the myeloperoxidase-mediated reaction. Control experiments were performed by omitting either myeloperoxidase or H202. After the incubation, ALP activities against trypsin and neutrophil elastase were measured as described below.

Effect of cis-platinum(II)diammine dichloride (cisplatinum) on the inhibitory activity of ALP ALP was reacted with cis-platinum essentially as described by Gonias et al. [23]. In brief, ALP was dissolved at a concentration of 1.4 #M in buffer (pH 7.4) containing 30 mM "Iris, 50 mM NaC1, 0.02% Triton X-100, and mixed with an equal volume of a solution of cis-platinum in 0.15 M NaC1, 1% mannitol. The concentrations of cis-plafinum in the mixture ranged from 52-1670/~M. After the mixture was incubated for 4 h at 37 o C, inhibitory activities of ALP against neutrophil elastase, chymotrypsin and trypsin were measured as described below, cis-Platinum was found not to interfere with the inhibitory activity measurements. The amount of platinum, bound to 15/xM ALP after an incubation with 3.3 mM cis-platinum, was assayed according to the method of Ayres and Meijer [24] and described in detail by Gonias et al. [23]. Binding of platinum to ALP was also measured after ALP (40/xM)

180 was previously incubated with iodoacetamide (0.15 M) in order to block possible free sulphhydryl groups.

Effect of second-domain specific monoclonal antibody on trypsin inhibitory activity of native ALP and recombinant second domain The ALP-specific monoclonal antibody produced by the mouse hybridoma clone 15 and purified from ascitic fluid was used in this study [2]. The specificity of MoAbl5 for the second domain of ALP was demonstrated by a positive response when the recombinant second domain was tested in a double-antibody sandwich immunosorbent assay using MoAbl5 as the first antibody and peroxidase-labeled anti-ALP rabbit immunoglobulin as the second antibody [2]. In addition, binding of MoAbl5 to recombinant second domain was observed by immunoblot analysis after sodium dodecyl sulphate gel electrophoresis in 16.5% acrylamide gels. Immunoblotting of gels was performed essentially as described by Towbin and Gordon [25]. The blot was incubated with MoAbl5, followed by incubation with rabbit anti-mouse IgG conjugated to alkaline phosphatase. Phosphatase activity was measured using 5bromo-4-chloro-3-indolyl phosphate and Nitro blue tetrazolium. Prestained molecular mass protein standard was used (BRL, Eggenstein, F.R.G.). To test the effect of MoAbl5 on the inhibitory activity of ALP, 18/~g active ALP was mixed with 500 /~g purified MoAbl5 antibody, in a final volume of 0.5 ml PBS. After an overnight incubation at 4 o C, dose-response trypsin inhibitory activity measurements were performed using N-a-benzoyl-Arg-pNA as described by Hoffmann et al., [21] except that the assay was not automated and was performed at 37 o C. Control experiments were performed by replacing MoAbl5 by purified ALP-specific monoclonal antibodies produced by hybridoma clone 5 and 31 (MoAb5 and MoAb31) [2]. Apparent inhibition constants (Ki,app) were calculated from the inhibition curves according to described methods [26,27]. The effect of MoAb15 on the trypsin inhibitory activity of recombinant second domain was studied by incubating 0.5 /~g recombinant second domain (---60 pmol) with increasing amounts of MoAb15 (0.1-0.5/zg antibody). Trypsin inhibitory activity was measured using the substrate benzoyl-Ile-Glu-Gly-Arg-pNA and the method described by Meckelein et al. [20]. Inhibitory activity measurements of ALP Inhibitory activity of ALP against neutrophil elastase was measured in rnicrotitre plates by mixing 100 #1 elastase (26 nM in PBS containing 0.1% gelatin: PBSgelatin) with 100/~1 inhibitor, diluted in 0.07 M Tris, 0.7 M NaC1, 0.1% gelatin (pH 8.3). After 30 rain at room temperature, 20 #1 of substrate (8 mM pyro-Glu-ProVal-pNA in dimethyl sulphoxide) were added [22]. The

microtitre plates were incubated at room temperature. Absorbance at 405 nm was measured at t = 0, 30 and 60 min using a Titertek Multiskan photometer (Flow Laboratories, Edinburgh, U.K.). Inhibitory activity of ALP against bovine pancreatic trypsin was measured in microtitre plates, based on the method described by Hoffmann et al. [21]. In the trypsin inhibition assay 50 #1 trypsin solution (70 nM in 0.625 M Tris, 25 mM CaC12, 0.1% gelatin, pH 7.8) were incubated for 30 min at room temperature with 25 /~1 inhibitor diluted, when required, in PBS-gelatin. Thereafter, 150 /LI substrate solution (2 mM N-a-benzoylArg-pNA in H20 ) were added [21]. The change in absorbance at 405 nm was measured as described above. Chymotrypsin inhibitory activity of ALP was determined as described by Delmar et al. [28]. In short, 275 /~1 a-chymotrypsin (20 nM in PBS-gelatin) were mixed with 275/~1 inhibitor diluted in 0.2 M Tris, 0.02 M CaC12, 0.1% gelatin (pH 7.8). After 10 min at room temperature, 50 /tl Suc(Ala)2ProPhe-pNA (6 mM in dimethyl sulphoxide) were added. The increase in absorbance was measured at 405 nm in a spectrophotometer using plastic disposable cuvettes. Results

Based on the assumption that the first domain of ALP inhibits trypsin and that the second domain inhibits elastase [7,8], we investigated whether ALP is capable of inhibiting trypsin and elastase simultaneously. As can be seen in Fig. 1, addition of elastase to trypsin-ALP complexes (molar trypsin/ALP ratio 1:4)

trypsin act.

elastaee act.

0.t4

-2

0.12 1.6 0.1

0.08

1.2

0.0S

0.8

0.04 0.4

0.02 0

0 1

2

3

4

elestase (molar excess Over ALP)

Fig. 1. Displacement of trypsin from native antileukoprotease (ALP) by the addition increasing a m o u n t s of neutrophil elastase. TrypsinA L P complexes were prepared by mixing trypsin and A L P at a molar ratio of approx. 1:4. Under this condition some free trypsin is still present which can be explained by the K i value of the complex (see legend Fig. 5). Elastase w a s added to the mixture at e l a s t a s e / A L P molar ratios ranging from 0.6 to 4.7. Enzyme activities are expressed as absorbance increase per rain at 405 n m (AA/min). The activity of trypsin in the absence of A L P was 0.158 AA/min. Final concentrations of trypsin and A L P in the trypsin inhibition assay were 80 and 280 nM, respectively. Trypsin activity ( × ) and elastase activity (D).

181 ALP inhibitory activity (% o f max) 100~1

80

60

40-

20-

0

,

o

2

i

,

, 8 myeloperoxidaee (nmol/I)

,

8

,

'

lO

Although the ALP molecule comprises two homologous domains of similar architecture [11], the second COOH-terminal domain has, in contrast to the first NH2-terminal domain, several methionine residues (Met 73, Met 82, Met 94 and Met 96) of which one is located in the proposed inhibitory active site (Leu72-Met73Leu74). The presence of this oxidation-susceptible residue in the inhibitory site of the second domain may give rise to a rapid oxidative inactivation of the activity of the second domain [29,30]. We therefore investigated the effect of the methionine-selective myeloperoxidaseoxidizing system on the inhibitory activity of ALP. As is

Fig. 2. The effect of human neutrophil myeloperoxidase/H202/C1system on ALP activity against elastase and trypsin. The ALP concentration during the incubation with myeloperoxidase was 3.3/~M. ALP inhibitory activities were calculated from the relative amount of enzyme inhibited and expressed relative to the activity of ALP which was not previously incubated with myeloperoxidase. H202 (213/tM) in the absence of myeloperoxidase had no effect on the inhibitory activity of ALP. Trypsin inhibition (*) and elastase inhibition (rn).

max)

a l a c t a c e act. (% of

'oi \ resulted in a dose-dependent release of active trypsin from the complex. At a molar elastase/ALP ratio of 1:1, more than half of the complexed and inhibited trypsin was released from the complex. In another experiment we also observed that an elastase-ALP complex, prepared by adding 0.45 nmol elastase to 0.3 nmol ALP, showed only 10% relative trypsin inhibition activity, compared to the activity of uncomplexed ALP when 0.15 nmol trypsin was added to the complex. The results of these experiments indicate that ALP inhibits either trypsin or elastase, and consequently seems to be unable to form an elastase-ALP-trypsin complex.

I

ol 0

10

20

30

40

60

70

6o

ALP (nmol/l)

trypein act. (% of max) 100i10604020-

ALP Inhibitory activity (% of max) 100'

0

o

so

loo

1so ~oo 2so ALP (nmol/I)

80 60

aoo

aso

40o

oflymotrypeln aot. (% of max) 100

40-

.oi\\

20

.....

0 600

10'00 16()0 cls-platlnum (micromol/I)

2000

Fig. 3. The effect of cis-platinum(II)diamminedichloride (cis-platinum) on ALP activity against elastase, chymotrypsin and trypsin. The ALP concentration in the mixture with cis-platinum was 0.7 #M. ALP inhibitory activities were calculated from the relative amount of enzyme inhibited and expressed relative to the activity of ALP which was not incubated with cis-platinum, cis-Platinum did not show any direct effect on the activity of enzymes used in the inhibitory assay measurements. Elastase inhibition (ta), trypsin inhibition ( × ) and chymotrypsin inhibition (*).

20 ~

~

~

o; o

lo

~

1~

40

60

00

ALP (nmol/l)

Fig. 4. Inhibition of elastase (12 nM), trypsin (16 nM) and chymotrypsin (9 nM) by ALP (*) and ALP incubated with 1670 ~tM cis-platinum (x). Enzyme activities are expressed as percentage of non-inhibited activities.

182 shown in Fig. 2, incubation of ALP with increasing amounts of human neutrophil myeloperoxidase in the presence of H202 and C1- results in a dose-dependent decrease of the ALP inhibitory activity against trypsin and elastase. Experiments were also performed with A L P and cis-platinum(II)diammine dichloride (cis-platinum). cis-Platinum is an anti-tumour agent able to bind covalently to nucleophilic groups in macromolecules [31]. In proteins, cysteine and methionine residues react readily with cis-platinum. Fig. 3 shows that treatment of ALP with increasing concentrations of cis-platinum results in a dose-dependent inactivation of the ALP inhibitory activity, irrespective of whether elastase, trypsin or chymotrypsin has been used in the inhibition assay. Enzyme inhibition curves of ALP and ALP incubated with 1670/~M cis-platinum are shown in Fig. 4. Platinum binding studies, performed at a cis-platinum concentration of 3.3 mM, revealed 3.6 + 1.1 (mean + S.D.; n = 4) mol of platinum bound to each mol of ALP. In one experiment, platinum binding to ALP, which was reacted with iodoacetamide prior to incubation with cis-platinum, was determined. This experiment revealed a similar binding ratio of platinum to ALP (3.9 mol platinum per mol ALP). It was observed that incubation of ALP with increasing amounts of ALP-specific monoclonal antibody, as produced by the mouse hybridoma clone 15 (MoAbl5) [2], resulted in a selective dose-dependent increase of the trypsin inhibitory activity of ALP. In contrast, the elastase and chymotrypsin inhibitory activities were not affected. Apparent inhibition constants of 19 and 0.4 nM were calculated from the trypsin inhibition curves obtained with ALP and ALP in the presence of excess MoAbl5 antibody showing maximal effect, respectively (Fig. 5).

trypsin Inhlb. act. (% of Initial set.}

225

~ 125 75

175

0

o.t 0.2 0.3 0.4 0.5 monoclonal antibody M o A b 1 5 (micrograms)

Fig. 6. The effect of monoclonal antibody MoAbl5 on the trypsin inhibitory activity of the recombinant COOH-terminal second domain of ALP. Concentrations of trypsin and second domain of ALP in the inhibition assay were 6 and 70 nM, respectively. The inhibitory activity was calculated from the relative amount of trypsin inhibited and expressed relative to activity of the second domain which was not incubated with monoclonalantibody. Incubation of two other ALP-specific monoclonal antibodies (MoAb5 and MoAb31) [2] did not result in any effect on the inhibitory activities of ALP. A similar dose-dependent increase in the affinity for trypsin was observed when the recombinant second domain of A L P was incubated with increasing amounts of M o A b l 5 (Fig. 6). MoAb5 and MoAb31 [2] did not show any effect. Under the test conditions used, the relative amount of trypsin inhibited by the recombinant second domain increased from 40 to 82% when up to 0.5/~g M o A b l 5 was added to 0.5/xg domain. The specificity of M o A b l 5 for the second domain was confirmed by analysis in a double-sandwich ELISA (data not shown) and by immunoblot analysis (Fig. 7).

trypsin act. (% Of max} IO0

8O

6O

4O

20

"~

0 0

20

,' 40

A L P or

60

80

tO0

120

ALP-antibody (nmol/I)

Fig. 5. Inhibition of trypsin (30 nM) by ALP (x) and by ALP previously incubated with monoclonal antibody MoAbl5 (ALP-antibody: *). Enzymeactivities are expressed as a percentage of non-inhibited activities. As calculated from these curves, apparent inhibition constants of 19 and 0.4 nM were obtained, respectively.

Fig. 7. Immunoblot showing binding of the ALP-specificmonoclonal antibody 'MoAbl5' to the second ALP domain. Lane 1, purified recombinant ALP [19]; lanes 2 and 3, recombinant second ALP domain [20]; lane 4, recombinant first ALP domain [20]; and lane M, molecular mass marker proteins. Additional bands in lane 1 are due to proteolyticdegradation during affinity purification of recombinant ALP using anhydrochymotrypsin.

183 Discussion

ALP consists of two domains (residues 1-56 and 57-107) [7-9], showing an internal sequence homology of 35%. The conserved amino acids include all the cysteine residues, of which eight are present in each domain [7,8]. Based on a slight homology with the active site of Kazal inhibitors [32] and the localization in the ALP molecule at which limited proteolytic cleavage can occur, several investigators speculate that trypsin binds to the first NH2-terminal domain, whereas elastase and chymotrypsin bind to the second domain [7,8,11,12]. Using X-ray crystallographic analysis Gri~tter et al. [11] showed that the ALP structure comprises two well-separated domains of almost identical architecture. It was also shown by these investigators that the reactive site loop of the second domain fits to the binding site of bovine chymotrypsin and possibly to that of human neutrophil elastase. In contrast to the assumption that elastase and chymotrypsin are inhibited by the second domain whereas trypsin is inhibited by the first domain, we found several lines of evidence that the inhibitory activities of ALP are all located in its second domain. When, as suggested, trypsin is inhibited by the first domain of ALP, the inhibitor should be able to inhibit elastase and trypsin simultaneously. However, we found, as can be seen in Fig. 1, that addition of elastase to trypsin-ALP complexes resulted in a displacement of trypsin from the inhibitor molecule. The ALP molecule is able to inhibit either trypsin or elastase, and a trypsin-ALPelastase complex is unlikely to be formed. The inability of ALP to inhibit elastase and trypsin at the same time was also supported by the observation that an ALPelastase complex was hardly able to inhibit trypsin. These results may be explained by assuming that trypsin and elastase are inhibited by the same domain, with which elastase is able to form a more stable complex than trypsin. The inhibition constants (Ki) of ALP for elastase and trypsin are 0.3 [33] and 19 nM (this study), respectively. An alternative explanation for the inability of ALP to form a trypsin-ALP-elastase complex may be the induction of large conformational changes in the first domain when elastase binds to the second domain, resulting in loss of affinity between trypsin and the first domain. However, the latter explanation is unlikely as the ALP chain is organized into two well-separated distinct domains [11]. The methionine residue at position 73 in the ALP molecule most likely forms part of the active inhibitory site [7,8,11]. Methionine at position 96 is probably also in close contact with residues of the enzyme inhibited [11]. The importance of these methionine residues in the inhibition activity of the second domain very likely explains the sensitivity of ALP to oxidation [18,34]. In the present study we observed that the elastase and

trypsin inhibitory activities of ALP did show a dose-dep e n d e n t susceptibility to purified neutrophil myeloperoxidase when incubated in the presence of hydrogenperoxide and chloride ion (Fig. 2). The myeloperoxidase/H202/C1- system is capable of oxidizing rather selectively methionine residues in proteins [29,35,36]. Similarly, incubation of ALP with increasing amounts of N-chlorosuccinimide, which is another methionine-selective oxidant [37,38], also resulted in a dose-dependent inactivation of the ALP activity as measured towards elastase, chymotrypsin and trypsin (unpublished observation). In addition, we also employed cis-platinum to study the importance of methionine residues in the inhibitor function of ALP. cis-Platinum is claimed to be extremely selective for functional methionine residues [30]. For example, it was found to be a selective modifier of the oxidation-sensitive methionine in the reactive site of al-proteinase inhibitor and of a methionine residue in a2-macroglobulin [23,39]. We found that reaction of ALP with increasing amounts of cis-platinum resulted in a concentration-dependent loss of the ALP activity (Figs. 3 and 4). It is very likely that the methionine residues in the second domain of ALP are the platinum-reactive amino acids. We found that 1 mol of ALP binds a maximum of 4 mol of platinum. This number equals the number of methionine residues in ALP. Other residues which may serve as platinum binding sites in proteins are cysteine and histidine [31,40]. However, alkylation by iodoacetamide of cysteine residues, which are unlikely to be present [11], did not result in a lower platinum binding to the ALP molecule. Moreover, histidine residues to which cis-platinum may bind, do not occur in the ALP sequence. Thus, the loss of inhibitory activity against trypsin, chymotrypsin and elastase is very likely due to binding of platinum to the methionine residues in the second domain of ALP. The results obtained in this study strongly support the idea that the trypsin, chymotrypsin and elastase inhibition sites are all located in the second domain of the ALP molecule. It has been shown recently that recombinant second domain, either expressed in E. coli [13] or in Saccharomyces cereoisiae [12] is a fully active elastase inhibitor. In addition, we observed in the present study that the second domain, as expressed in E. coli, also shows activity, although weakly, against trypsin. The low potency of the separate second domain to inhibit trypsin suggests that this activity is affected negatively by the absence of the NH2-terminal domain, or that, alternatively, the recombinant product is not in its optimal active conformation to inhibit trypsin. The trypsin inhibitory activity of this recombinant domain could be potentiated by adding a second domain-specific monoclonal antibody (Fig. 6). This antibody reveals a similar effect on the intact ALP molecule (Fig. 5). Therefore, the results obtained from experiments per-

184 f o r m e d with the r e c o m b i n a n t second d o m a i n a n d the m o n o c l o n a l a n t i b o d y s u p p l y a d d i t i o n a l evidence t h a t the trypsin i n h i b i t o r y activity o f A L P is l o c a t e d in its second d o m a i n . T h e m e c h a n i s m b y which the i n h i b i t o r y activity against trypsin is p o t e n t i a t e d when the i n h i b i t o r is in c o m p l e x with the m o n o c l o n a l a n t i b o d y is unknown. O n e e x p l a n a t i o n might b e that a d d i t i o n a l residues in the i n h i b i t o r molecule or in the a n t i b o d y molecule b e c o m e involved in the t r y p s i n - i n h i b i t o r interactions, resulting in an increase of the affinity between the e n z y m e a n d inhibitor. Based o n the results presented, it is n o t clear w h a t functional activity resides in the N H 2 - t e r m i n a l d o m a i n of A L P . V e r y recently it has b e e n o b s e r v e d that, at very high enzyme a n d i n h i b i t o r c o n c e n t r a t i o n s (6 /~M or more), each molecule of A L P is a b l e to inhibit two molecules of trypsin, c h y m o t r y p s i n or c a t h e p s i n G ( c h y m o t r y p s i n - l i k e enzyme), b u t o n l y one m o l e c u l e of elastase ( p e r s o n a l c o m m u n i c a t i o n f r o m J.G. Bieth, U n i versit6 Louis Pasteur de Strasbourg, Illkirch, France). This w o u l d i n d i c a t e that the first d o m a i n is a b l e to b i n d trypsin, c h y m o t r y p s i n a n d c a t h e p s i n G at a very low affinity, which is unlikely to b e of a n y p h y s i o l o g i c a l importance. I n conclusion, the o b t a i n e d results p e r f o r m e d with A L P and its r e c o m b i n a n t C O O H - t e r m i n a l d o m a i n reveal strong evidence that the i n h i b i t o r y activities o f native A L P against trypsin, c h y m o t r y p s i n a n d elastase are represented b y its s e c o n d d o m a i n . T h e m e t h i o n i n e residues in this d o m a i n seem to p l a y an essential role in the i n t e r a c t i o n of A L P with proteinases, m a k i n g the i n h i b i t o r sensitive to oxidation. R e c o m b i n a n t A L P [12,19], r e c o m b i n a n t s e c o n d d o m a i n [12,13,20] o r o x i d a t i o n - r e s i s t e n t m u t a n t s m a y b e very useful as a t h e r a p e u t i c agent in the m a n a g e m e n t of p u l m o n a r y e m p h y s e m a . This disease is t h o u g h t to develop b e c a u s e of e l a s t a s e - m e d i a t e d d e s t r u c t i o n of connective tissue c o m p o n e n t s [41]. A L P is f o u n d to be a very p o t e n t i n h i b i t o r of e l a s t a s e - m e d i a t e d d e g r a d a t i o n of extracellular m a t r i x m a c r o m o l e c u l e s [42-44]. M o r e over, small inhibitors m a y p e n e t r a t e b e t t e r into pericellular m i c r o - e n v i r o n m e n t s to inhibit e l a s t a s e - m e d i a t e d m a t r i x d e g r a d a t i o n b y n e u t r o p h i l s [44,45].

Acknowledgements The authors t h a n k Miss A. M i d d e r h a m a n d Miss E.A. van d e r K w a s t for p r e p a r i n g the m a n u s c r i p t .

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Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain.

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. T...
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