Eur. J. Biochem. 202,1223-1230 (1991)

c) FEBS 1991

Mercurial activation of human polymorphonuclear leucocyte procollagenase Jorg BLASER, Vera KNAUPER, Anja OSTHUES, Heinz REINKE and Harald TSCHESCHE University of Bielefeld, Faculty of Chemistry, Department of Biochemistry, Bielefeld, Federal Republic of Germany (Received June 26, 1991) - EJB 91 0833

The mechanism of human polymorphonuclear leucocyte (PMNL) procollagenase activation by HgCl, was investigated by kinetic and sequence analysis of the reaction products. HgClz activated PMNL procollagenase by intramolecular autoproteolytic cleavage of the Asn53 - Val54 peptide bond to generate a collagenase species of M , 65000, which was immediately converted into a second intermediate collagenase form by further autoproteolytic cleavage of the Asp64 - Met65 peptide bond within the propeptide domain. This intermediate form (Met65 N-terminus) reached maximum concentrations after 45 min and displayed only about 40% of the maximum available enzymatic activity. Final activation was obtained after autoproteolytic cleavage of either Phe79 - Met80 or Met80 - Leu81 peptide bonds. Furthermore, activation in the presence of TIMP-1 did not suppress the intramolecular autoproteolytic cleavage of the Asn53 - Val54 peptide bond. Complete inhibition of further autoproteolytic decay of the enzyme or generated peptides was observed, which was obviously due to complex formation between the intermediate collagenase form (Val54 N-terminus) and inhibitor, which was visualized using the Western blot technique. Thus PMNL procollagenase activation by HgCl, followed a three-step activation mechanism which is entirely different from the known activation mechanisms of the fibroblast matrix metalloproteinases.

The degradation of interstitial and basement membrane collagens by polymorphonuclear leucocytes (neutrophils) is initiated by two specific metalloproteinases: PMNL collagenase and PMNL gelatinase. The primary structure of these enzymes has recently been elucidated by cDNA analysis [l, 21 and it has been shown that they are distinct gene products [l -41, sharing homology with other matrix metalloproteinases [5- 111 (for review see [12]). As these metalloproteinases are secreted as latent precursors their activation is the most important step during initiation of collagenolysis. It has been reported that the activation of latent PMNL collagenase can occur by several mechanisms. This includes proteolysis by various proteases [3], exposure to mercurials [13,14] or gold(1) compounds [I 51, and oxidative activation or 'autoactivation' [16]. Though mercurials are unable to cleave peptide bonds themselves they have often been used as valuable activating compounds. Proteolytic cleavages by added proteases, such as trypsin, cathepsin G or others, on the protein core of the PMN L procollagenase can be excluded by using mercurials. This enables the investigation of the autoproteolytic activation process initiated by mercurials. We demonstrate that the activation of PMNL procollagenases ( M , 85 000) by mercurial compounds is associated with a reduction in the molecular Correspondenre to H. Tschesche, Universitat Bielefeld, Fakultat fur Chemie, Abteilung Biochemie, Postfach 8640, Universitatsstrasse, W-4800 Biclefeld 1, Federal Republic of Germany Abhreviarions. APMA, (4-ammopheny1)mercuric acetate; CIHgBzOH, 4-chloromercuribenzoic, acid; PVDF, poly(viny1idene difluoride); OHBzHgOH, 4-hydrnxymercuribenzoic acid; PMNL, polymorphonuclear leucocytes; RP-HPLC, reversed-phase high-performance liquid chromatography; TIMP-1, tissue inhibitor of metalloproteinases-1 ; Dnp, dinitrophenyl. Enzymes. Alkaline phosphatase (EC 3.1.3.1); human PMNL collagenasc (EC 3.4.24.7).

mass of the enzyme; there is evidence for autoproteolytic cleavage, in contrast to a recent report [14]. The reaction products have been further investigated by N-terminal sequence determination, revealing a three-step activation mechanism.

METHODS Purification of PMNL procollagenase, activation and enzyme assay PMNL procollagenase was purified as recently published [3]. Proenzyme activation was achieved by incubation with different mercurial compounds at 37°C (for further details see Fig. 1). Collagenase activity was determined by proteolytic degradation of the synthetic octapeptide (Dnp-Pro-Gln-GlyIle-Ala-Gly-Gln-D-Arg-OH) as described by Masui [ 171. Alternatively, the degradation of soluble type I collagen at 25 "C was shown by SDS/PAGE. Purification of TIMP-1 Human tissue inhibitor of metalloproteinases-1 (TIMP-1) was purified from rheumatoid synovial fluid by a modified method according to Wilhelm et al. [ 2 ] . Inhibition by TIMP-1 PMNL procollagenase was activated for 2 h using 1 mM HgC1,. The activated enzyme was incubated for 15 min with varying amounts of human TIMP-1. The remaining collagenase activit) was determined by the degradation of the synthetic octapeptide (Dap-Pro-Gln-Gly-Ile-Ala-Gly-Gln-~Arg-0 H).

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Fig. 1. (A) Concentration-dependentactivation of PMNL procollagenase by HgC12 and (B) requirement of the continued presence of HgClz for complete PMNL procollagenase activation. (A) PMNL procollagenase ( 5 pg) was incubated with 1 mM (W), 100 pM ( 0 )and 10 pM (A) HgCI, at 37'C. The collagenolytic activity was measured against the Dnp-octapeptide for 30 min at 37°C [17]. (----) Activity of PMNL procollagenase in the presence of buffer. (B) PMNL procollagenase (50 pg) was incubated with 50 pM HgClz for 30 min at 37°C. HgClz was removed from 50% of the reaction mixture by desaiting using a Sephadex G-10 column equilibrated with 20 mM Tris/HCI pH 7.5, 0.3 M NaCI, 5 m M CaCIZ.The HgClz-freecollagenase was further incubated at 37 "C and the collagenolytic activity was time-dependently monitored using the Dnp-octapeptide assay [17]. ( 0 )Collagenase in the presence of HgCI2; (A)collagenase with HgClz removed; (----) PMNL procollagenase in the presence of buffer.

Kinetic analysis of HgCl,-induced activation of PMNL procollagenase and separation of the reaction products by RP-HPLC PMNL procollagenase was activated for between 5 min and 2 h at 37°C using 1 mM HgCI2. The activated enzyme was subjected to a Bakerbond wide-pore C I 8 column (4.9 x 250 mm). The separations of the reaction products were performed at a constant flow rate of 0.8 ml/min using a linear gradient of 0 - 80% acetonitrile. Peptides and proteins were detected at 214 nm, collected, lyophilized and subjected to automated amino acid sequence determination. Kinetic analysis of HgC12-inducedactivation of PMNL procollagenase in the presence of equimolar amounts of TIMP-1 PMNL procollagenase was preincubated for 5 min with equirnolar amounts of human TIMP-1. Activation of the protein mixture with HgC12was performed as described +hove SDSf PAGE

SDSjPAGE was performed arcording to the method of Laemmli [18]. The proteins we& visualized by silver staining [I 91.

SDS-stable TIMP-l/collagenase complexes PMNL procollagenase ( 5 pg) was either preactivated by treatment with 1 mM HgC12 at 37°C for 2 h followed by the addition of TIMP-I (1.6 pg) or directly activated in the presence of the inhibitor. Laemmli sample buffer (unreduced) was added to the reaction mixture giving a final concentration of 1% SDS. The samples were not heated and immediately applied to electrophoresis. Western blotting Antigens were separated by SDSjPAGE and blotted onto a PVDF membrane using the Biometra fast blot system. To minimize unspecific staining the membrane was incubated in 3% (massjvol.) dry milk in buffer A (50 mM TrisjHCI pH 7.9; 0.15 M NaCI, 0.05%, mass/vol., Tween 20) for 30 min. Specific antisera (either anti-collagenase or TIMP-1 immunglobulins) were added using concentrations of 1 pg/ml. After 2% thehlot membrane was extensively rinsed in buffer A to remove un6ound immunglobulins. Antigen-antibody complexes were detectd by the reaction with goat anti-(rabbit IgG) - alkaline-phosphatase conjugate (Boehringer Mannheim) diluted 1 : 10000 for 1 h. Visualization was performed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as alkaline phosphatase substrates. Sequence determination

Preparation of specific antisera Rabbits were immunized with approximately 400 pg purified antigen (either human PMNL collagenase or human TIMP-1) as rec mtly published [20].

Amino-terminal sequence determinations were performed as recently published [4] using a 1,iicrosequencer (Modell 810, Knauer, Berlin, FRG) and a modified method of Hunkapiller

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mol TIMP-1 / mol collagenase Fig. 2. Inactivation of active PMNL collagenase by TIMP-1. PMNL collagenase (500 ng). preactivated with 1 mM HgCIZ,was mixed with increasing amounts of human TIMP-1. Activity remaining was assessed using the Dnp-octapeptide assay [ 171

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Fig. 4. Activation of PMNL procollagenase by mercurial compounds. PMNL procollagenase ( 5 pg) incubated with different mercurials (1 mM) for 2 h at 37°C were subjected to SDSjPAGE (10% gel) without reduction and silver-stained. Lane 1, procollagenase; lanc 2, HgCl,-activated; lane 3, rnersalylic-acid-activated; lane 4, APMAactivated; lane 5, CIHgBzOH-activated; lane 6 , OHBzHgOH-activated.

8

20.5 14.4 Fig. 3. Demonstration of PMNL collagenase-TIMP-1 complexes. Recognition of PMNL collagenase and PMNL-collagenase-TIMP-I complexes using anti(T1MP-I) IgG (lanes 1-4) and anti(P>n’”L cc:lagenase) TgG (lanes 5 - 8) on Western blotting. Lanes 1 dnd 3, PMNL procollagenasc; lanes 2 and 6 , PMN L procollagenase activated by HgCI, in the presence of TIMP-1; lanes 3 and 7, PMNL procollagenasc, preactivated by HgCI, and inhibited by the addition of TIMP-1; lanes 4 and 8, PMNL procollagenase activated by HgCI2 in the absence of TIMP-1.

RESULTS Human PMNL procollagenase activation by mercurial compounds Incubation of human PMNL procollagenase with HgC12 at concentrations of 10 FM, 100 pM and 1 mM showed the

activation of the proenzyme in a gradual and concentrationdependent fashion (Fig. 1A). About 100% activation was achieved by treating the proenzyme with 100 pM HgClz for 3.5 h. PMNL procollagenase was activated by 10 pM HgClz after 4 h to about 40% of the maximum activity. In contrast, pretreatment of PMNL procollagenase with higher concentrations of HgCI2 (1 mM) resulted in faster generation of collagenolytic activity, but full activation was not achieved and the enzymatic activity decreased markedly after a 1.5-h incubation, which was probably due to fragmentation of the active enzyme (unpublished results). The dependency on the presence of the activating mercurial agent was further investigated. Therefore partial activation of the proenzyme by 50 pM HgC12 was achieved after a 30-min incubation. After removal of HgCI2 by desalting only 50% of the collagenolytic activity was achieved as compared to those aliquots of the enzyme which remained in the presence of HgClZ (Fig. l B ) . Thus, maximum activation of the enzyme was dependent on the presence of the activating mercurial compound. Furthermore, the proenzyme was activated to different extends by (4aminopheny1)mercuric acetate (APMA), 4-chloroinercuribLnzoic acid (CIHgBzOH), 4-hydroxymercuribenzoic acid (OHBzh SOH) and mersalylic acid (Fig. 4). Inactivation by TIMP-i Active human PMNL cui: igenase was inhibited by the specific tissue inhibitor of metall, proteinases, TIMP-1. Inactivation was approximately linear up to 90% inactivation. By extrapolation it was calculated that T‘WP-1 and PMNL collagenase react by formation of an equimolar enzyme-inhibitor complex (Fig. 2). The complex between TIMP-1 and active PMNL collagenase was partially stable to SDSjPAGE and showed an apparent M , of 92000 and was visualized by West-

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YO Acetonitrile Fig. 5. Time-dependent activation of PMNL procollagenase by HgC12. PMNL procollagenase (SO gg) was incubated with Z mM HgClz over a period of 2 h. (A) SDSjPAGE analysis of generated reaction products. Lane 1, molecular mass markers (Mrx lane 2, starting material PMNL procollagenase; lanes 3 -7, after exposure to 1 mM HgClz for 5 min, 15 min, 30 min, 60 min and 120 min respectively. (B) Separation of the reaction products generated during HgCIz activation of PMNL procollagenase by reversed-phase HPLC (schematic drawing of relevant signals). Profile 1, procollagenase; profiles 2 - 6, after exposure to 1 mM HgClz for S min, 15 min, 30 min, 60 min and 120 min, respectively. Peaks: a and b, propeptide fragments (residues Phel - AsnS3 ; the slightly different retention times are due to the variable glycosylation degree); c , propeptide fragment (residues Val54 - Asp64); d. autoproteolytic degradation product of peptides a and b (residues Gln22 AsnS3); e, autoproteolytic degradation product of peptides a and b (residues Tyrl7-Asn21); f, autoproteolytic degradation of the peptide d (residues Gln22 - Lys42 or Gln22 - Arg48; N-terminus Gln22), g, autoproteolytic degradation of the peptide d (residues Gln22 - Arg48 or GI1122 - Lys42; N-terminus Gln22); h, autoproteolytic degradation of the peptide d (residues Phe49 -AsnS3); i, autoproteolytic degradation of the peptide d (residues Leu43-AsnS3); j, autoproteolytic degradation of the peptides a and b (residues PheZ -Aspl6). ern blottting using specific anti-collagenase [20] and antiTIMP-1 immunglobulins (Fig. 3). Complete dissociation of the complex was observed upon heating or reduction (not shown). Autoproteolytic processing of human PMNL procollagenase induced by mercurial compounds

The activation of PMNL procollagenase ( M r 85 000) by different mercurials was accompanied by processing to a lowmolecular-mass enzyme ( M , 64000) as shown in Fig. 4, whilst the molecular mass of the latent control remained unchanged, and no autoactivation was observed. About 90% of the proenzyme was converted into active PMNL collagenase ( M r64000) by HgC12,APMA o r CIHgBzOH, which correlated well with the enzymatic activity generated. In contrast, OHBzHgOH and mersalylic acid were less effective in reducing the molecular mass and activating the proenzyme. The processed PMNL collagenase was analyzed by SDSjPAGE to detect short-lived intermediates of the enzyme. As seen in Fig. 5A, we were not able to identify intermediates using this method. The molecular mass reduction of the proenzyme induced by

organomercurial compounds was inhibited by EDTA, 1,lophenanthroline or lanthanide ions (not shown), giving evidence for autoproteolytic cleavage. In contrast, activation of purified PMNL procollagenase by HgC12 in the presence of TIMP-1 did not prevent molecular mass reduction. Comparison of the molecular masses of PMNL collagenase processed in the presence and absence of TIMP-1 (Fig. 6) showed that at least one short-lived intermediate enzyme form was generated. To confirm this, detailed kinetic and N-terminal sequence analyses were performed, and revealed that a n intermediate enzyme form was generated by an intramolecular selfcleavage process, which was followed by complete activation of the enzyme as a result of two further autoproteolytic processings (see below). Kinetic analysis of HgC12-inducedactivation of PMNL procollagenase

The activation of PMNL procollagenase induced by HgC12 was investigated by RP-HPLC, which allowed the separation of the reaction products followed by N-terminal sequence determination. Kinetic analysis of the reaction prod-

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boxyl end of the highly conserved region consisting of the sequence PRCGVPD, which contains the unpaired Cys71 residue of PMNL procollagenase.

Kinetic analysis of HgCI,-induced activation of PMNL procollagenase in the presence of human TIMP-1

94

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Fig. 6. Comparison of the molecular masses of different collagenase forms. PMNL procollagenase (50 pg) was activated by HgCI2 in the presence and absence of TIMP-1. The reaction products were purified by reversed-phase HPLC and subjected to SDSjPAGE and visualized by silver staining. Lane 1, molecular mass markers ( M , x lane 2, PMNL procollagenase; lane 3, processed in the presence of TIMP-1 (Val54 N-terminus); lane 4, processed in the absence of TIMP-1 (Met80 or Leu81 N-terminus).

ucts demonstrated the time-dependent generation of various peptides (Fig. 5B), which were the result of the autoproteolytic cleavage of the enzyme and further cleavage of the peptides liberated. After a 5-min activation by HgC12, the direct loss of two peptides was observed, which showed the N-terminal sequence of the proenzyme. The liberation of these peptides was the result of the formation of an intermediate collagenase form that was produced via autoproteolytic cleavage of the Asn53 -Val54 peptide bond within the propeptide domain. Since this intermediate form (Val54 N-terminus) was unstable in the presence of the activating mercurial compound, isolation and N-terminal sequence determination was only possible by suppressing further autoproteolytic decay using human TIMP-1. Otherwise, the immediate autoproteolytic release of a third peptide was noticed within 5 min. It was generated by autoproteolytic cleavage of the Asp64 - Met65 peptide bond, four amino acid residues preceding the conserved PRCGVPD sequence motif. The second intermediate form (Met65 N-terminus) was quite stable and its N-terminal sequence was obtained after a 15-min incubation and reached maximum concentrations after a 45-min activation. This collagenase form (Met65 N-terminus) displayed only 40% maximum enzymatic activity. Fully active PMNL collagenase was subsequently generated during prolonged incubation and two further peptides were released by cleavage of either the Phe79 - Met80 or Met80 - Leu81 peptide bond. PMNL collagenase activation followed a three-step activation mechanism. The determined N-termini of fully active PMNL collagenase corresponded to ,the homologous activation site(s) of human fibroblast collagenase, stromelysin and M,-72 000 gelatinase (Fig. 7) [22-251. This site is adjacent to the car-

Activation of PMNL procollagenase by HgC12 in the presence of TIMP-1 did not prevent the release of the two Nterminal peptides generated by cleavage of the Am53 -Val54 peptide bond by an intramolecular autoproteolytic process as earlier proposed for other matrix metalloproteinases [22, 24, 261. Since both peptides showed identical N-termini and the intermediate enzyme form a single N-terminus, we have evidence that these peptides differ only in their carbohydrate content. Effective inhibition of further processing was observed in the presence of TIMP-1, which was the result of complex formation between an intermediate collagenase form and the inhibitor, demonstrated here by Western blotting (Fig. 3).

ProcessingoftheN-terminal propeptides (residues 1 - 53) Mercurial-induced activation of PMNL procollagenase was accompanied by autoproteolytic cleavage of the single Asn53 - Val54 peptide bond as the primary event. However, two, instead of one, peptides were released from the propeptide domain. They showed slightly different retention times when analyzed by RP-HPLC. Both obviously differed in their degree of glycosylation because their N- and C-termini were identical and corresponded to residues 1 - 53 of the proenzyme. This propeptide fragment contains two carbohydrate attachment sites as recently elucidated by cDNA cloning [I]. Subsequently, these different glycopeptides were autoproteolytically degraded in a time-dependent fashion by cleavage of the Asp15 -Tyrl6 and the Asn21 -Gln22 peptide bonds (Fig. 8). Thus, three additional peptides were generated as a consequence of this autoproteolytic processing, which was catalyzed by the enzyme and inhibited by TIMP-1. The remaining larger propeptide fragment (Gln22 N-terminus) was hydrolyzed between either the residues Lys42 - Leu43 or Arg48 - Phe49 and resulted in the concomitant appearance of four additional peptides (Fig. 8). The autoproteolytic action of human PMNL collagenase on its own propeptide domain revealed a rather broad specificity of the bonds cleaved. Hydrolysis occurred at the Nterminal site of hydrophobic and non-polar amino acid residues, which could be elucidated as a characteristic feature of the bonds cleaved. DISCUSSION The results of the present study demonstrate that highly purified human PMNL procollagenase ( M , 85000) was activated by mercurial compounds with concomitant reduction of molecular mass by a three-step activation mechanism. This is unique among the known activation mechanisms of different members of the matrix metalloproteinase family. An early report by Sorsa and coworkers that the activation of latent PMNL collagenase was induced without change of the molecular mass of the enzyme could not be confirmed [14]. In our opinion this was probably due to the fact that the authors may have used an N-terminally truncated enzyme form during their activation experiments. Their collagenase form might

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Fig. 7. (A) Determined autoproteolytic cleavage sites of PMNL collagenase during mercurial activation and (B) comparison of the intermediates obtained during mercurial activation of different matrix metalloproteinases. (A) Cleavage sites are indicated in the propeptide sequence of the procnzymc by arrows. (B) Rcvealed cleavage sites are indicated by arrows. [A] PMNL collagenase (this paper); [B] fibroblast collagcnase [22]; [C] fibroblast collagcnase [23]; [D] fibroblast stromelysin [24]; [El M,-72000 gelatinase [25];[F] Mr-92000gelatinase [Z].

have been our second intermediate form (Met65 N-terminus) and it would be extremely difficult or impossible to determine a molecular mass reduction induced by mercurials. Our experiments showed a parallel increase in enzymatic activity accompanied by a molecular mass reduction of PMNL collagenase during activation, indicating that autoproteolytic cleavage was the most important step. The transient appearance of two intermediate enzyme forms was demonstrated, resulting in the conversion into the final, active form. The Asn53 -Val54 peptide bond of the propeptide domain was hydrolyzed by a rapid intramolecular autoproteolytic step, which was concentration-independent (not shown) and unaffected by the specific matrix metalloproteinase inhibitor TIMP-1. This intermediate form (Val54 N-terminus) showed a high rate of decay and was immediately converted to the second intermediate enzyme form by autoprpteolytic cleavage of the Asp64 - Met65 peptide bond. This second intermediate (Met65 N-terminus) reached highest concentrations in the reaction mixture within 45 mir, and displayed only about 40% of the maximum possible enzymatic activity. In conclusion, final activation was obtai;:ed after cleavage of either Phe79 Met80 or Met80 - Leu81 peptide bonds, thus demonstrating that the complete reinoval of the propeptide domain was essential for obtaining 100% enzymatic activity. There was no evidence for C-terminal truncation of the enzyme during mercurial activation of PMNL procollagenase. The observed

autoproteolytic processings led to a stepwise N-terminal truncation of PMNL collagenase without any apparent time lag, as demonstrated by RP-HPLC. In contrast, fibroblast collagenase, stromelysin and M,72000 gelatinase attained maximal collagenolytic activity prior to complete conversion to the stable, lower-molecularmass form. This is consistent with a conformationally rearranged, unstable and active enzyme species. A time lag was observed between the generation of enzymatic activity and autoproteolytic processing of the propeptide domain [22,24 271, which is entirely different from the behaviour of the PMNL homologue. Fibroblast collagenase and stromelysin were autaproteolytically processed by a two-step activation mechanism [22 - 241. In contrast, M,-72000 gelatinase followed a single-step activation mechanism [25], which indicates that the homologous matrix metalloproteinases follow distinct activation mechanisms to generate enzymatic activity. This might be due to their variable substrate specificities or the different conformational arrangement of the propeptidc within the active site of each enzyme during the autoproteolytic cleavage process. The primary cleavage site of PMNL collagenase (Am53 Va154) differed from the corresponding autoproteolytic processing sites of the homologous fibroblast matrix metalloproteinases, collagenase and stromelysin [22 - 241. A common feature of the investigated activation mechanism of fibroblast

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collagenase and stromelysin was the appearance of an intermediate enzyme form, which was processed 3 - 6 amino acid residues preceding the PRCGVPD sequence motif. PMNL collagenase was autoproteolytically cleaved at a similar position, generating the second intermediate form (Met65 Nterminus) (Fig. 7). Thus, in our opinion, it might be possible that the homologous proenzymes all follow a three-step activation mechanism as proposed for the PMNL collagenase, since the final autoproteolytic cleavage site(s) corresponded exactly to the homologous site(s) of fibroblast collagenase, stromelysin and MI-72000 gelatinase. To our knowledge no detailed investigations of matrix metalloproi.einase activation mechanisms were carried out in the presence of TIMP-1, followed by N-terminal sequence determination of possibly generated, very short-lived intermediate enzyme forms. Therefore, it might be possible that different investigators failed to detect enzyme forms which show a high rate of decay. Our present results showed that the presence of mercurials during the activation process of PMNL collagenase was essential for generating maximum enzymatic activity. It has been reported previously that both N-terminally processed PMNI, collagenase forms (Val54 and Met65 N-terminus) described here were still latent and exhibited no proteolytic activity against natural or synthetic substrates when measured in the absence of mercurial compounds [3, 281. The N-terminal truncation of the propeptide domain by autoproteolytic loss of 53 or 64 amino acid residues was insufficient to generate proteolytic active PMNL collagenase. This is in accordance with the results obtained during trypsin activation and elastase processing [3]. Thus, a short ‘propeptide fragment’ of only 26 or 15 residues might remain anchored to the integral activesite zinc preventing a conformational rearrangement and preserving latency. In contrast, both N-terminal truncated PMNL collagenase forms (Val54 and Met65 N-terminus) were autoproteolytically active in the presence of mercurial com-

48

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pounds. This might indicate that a competition reaction between the integral acitve-site zinc and the activating mercurials for binding Cys71 of the propeptide takes place. Binding of mercurials to the Cys71 residue would induce a conformational rearrangement and exposure of the propeptide domain to the active site of the enzyme, resulting in concomitant autoproteolytic processing. Recently, site-directed mutagenesis experiments using transin and human fibroblast cDNA clones showed, that the exchange of either Pro88, Pro93 (transin) or Cys73 (collagenase) in the strongly conserved PRCGVPD sequence motif led to ‘autoactive’ low-M, enzyme species 129, 301, indicating that a correct folding of the propeptide is required for latency. In correlation with the data obtained during mercurial and proteolytic activation of PMNL collagenase, the free Cys71 of the propeptide domain has to be removed from the coordination sphere of the integral active-site zinc by cleavage of either Arg70-Cys71 [3], Phe79 - Met80, MetSO- Leu81 or Leu81 -Thr82 peptide bonds at least. In conclusion, mercurial activation of PMNL procollagenase follows the rules of the ‘cysteine switch activation mechanism’, which comprises the concomitant removal of the conserved cysteine residue of the PRCGVPD sequence motif out of thc coordination sphere of the integral acitve-site zinc by autoproteolytic or proteolytic cleavage [3, 31 -331. The nature of human PMNL collagenase and TIMP-1 interaction was investigated hy assessment of enzymatic activity and SDS/PAGE. No komplex formation between PMNL procollagenase and inhibitor took place, which is in accordance to earlier published data on the fibroblast enzyme [34]. The conversion of PMNL procollagenase to the lowermolecular-mass intermediate form (Val54 N-terminus) could not be inhibited by the presence of equimolar amounts of TIMP-1, indicating that complex formation was only possible after N-terminal processing of the proenzyme. Only N-ter-

1230 minally truncated PMNL collagenase forms (Val54 and Met80 or Leu81 N-termini) reacted by enzyme-inhibitor complex formation, showing M , of 92000 and 94000, indicating a 1 : 1 stoichiometry. However, it was recently shown by DeClerck and coworkers that recombinant TIMP-2 blocked the activation of human fibroblast procollagenase by mercurials. This was due to the formation of an SDS-stable TIMP-2/procollagenase complex probably with a 2: 1 stoichiometry [35]. It remains to be seen whether TIMP-1 forms complexes with conformationally rearranged procollagenase when a twofold molar excess of inhibitor is used. Both PMNL collagenase TIMP-1 complexes failed to degrade natural or synthetic substrates, the enzymatic activity being completely blocked. The binding site of the inhibitor to N-terminally truncated PMNL collagenase forms must be entirely different from the binding site of TIMP-1 to purified Mr-92000 progelatinase of simian virus 40 transformed lung fibroblasts [2]. Whereas the Mr-92000-gelatinase- TIMP-1 complex displayed enzymatic activity against natural or synthetic substrates, when activated by mercurial compounds, both PMNL collagenase-TIMP-1 complexes were inactive, demonstrating the different nature of the enzyme inhibitor interaction. This work was supported by the Deutsche ForschungJgemeinschuft, special research programme SFB 223 project B2. The skilful assislance of K. Etzold and S. Rottmann is greatfully acknowledged. The authors wish to thank Mrs G. Delany for linguistic advice.

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