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J. Biol. Chem., Vol. 276, Issue 28, 26030-26035, July 13, 2001

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Maturation of Pseudomonas aeruginosa Elastase

FORMATION OF THE DISULFIDE BONDS^*

Peter Braun, Corrine Ockhuijsen, Elaine Eppens^§, Margot Koster, Wilbert Bitter^¶, and Jan Tommassen

From the Department of Molecular Cell Biology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands

Received for publication, August 7, 2000, and in revised form, May 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The opportunistic pathogen Pseudomonas aeruginosa secretes many proteins into the extracellular medium. The secreted proteins are synthesized in the cytoplasm and have to pass both membranes of the cell envelope. Four main pathways for the secretion of proteins, usually referred to as the type I, II, III, and the autotransporter pathway, have been identified in P. aeruginosa (1-4). The majority of the exoproteins characterized is secreted via the type II pathway, also referred to as the general secretory pathway. In addition to elastase, which is the most abundant secreted protein, lipase, alkaline phosphatase, exotoxin A, two phospholipases C, the staphylolytic protease LasA, the chitin-binding protein CbpD, and a putative aminopeptidase are secreted via this pathway (2, 5-7).

Proteins secreted via the type I or type III pathways are translocated across the two membranes of the cell envelope in a single step, without a periplasmic intermediate. In contrast, the type II pathway is a two-step mechanism. The first step is the translocation across the inner membrane, which is mediated by the Sec machinery. In the periplasm, which contains chaperones and folding catalysts, the exoproteins fold into a (near-)native conformation (8). For several proteins that are secreted via a type II mechanism (9-11), including elastase of P. aeruginosa (12, 13), it has been demonstrated that folding in the periplasm is essential for the subsequent translocation across the outer membrane to occur. In P. aeruginosa, the translocation of the periplasmic intermediates across the outer membrane is mediated by a machinery composed of at least 12 proteins, encoded by the xcp genes (for a review, see Ref. 2).

To investigate the biogenesis of proteins secreted via the type II pathway of P. aeruginosa, we have chosen elastase as a model. This metalloprotease, which is encoded by the lasB gene (14), is produced as a preproprotein. The pre- part is the signal peptide, which directs the translocation of the proenzyme across the inner membrane (15). The propeptide is essential for the folding of elastase in the periplasm (12, 13), and this folding allows for further processing of the proenzyme by autoproteolytic cleavage (16). The propeptide remains noncovalently associated with the mature elastase (15) and inhibits further proteolytic activity of the enzyme (17). Subsequently, the propeptide-enzyme complex is secreted and dissociates during or after translocation across the outer membrane (6, 18). Dissociation of the propeptide-enzyme complex is a well coordinated process, and a host-specific factor is required to induce this event (19). The propeptide is finally degraded by an extracellular protease (6, 18), probably elastase itself.

Proteins secreted via a type II pathway can acquire disulfide bonds, which are formed in the oxidizing environment of the periplasm by the presence of the Dsb system (for a review, see Ref. 20). The propeptide of elastase does not contain any cysteines, whereas the mature polypeptide contains four of them, which together form two disulfide bonds in the folded enzyme (21). Both bonds are not localized in close proximity of the active center of the protein. One disulfide bond, between Cys-30 and Cys-57, is located in the N-terminal part of the mature enzyme and connects two -strands. The other disulfide bond, between Cys-270 and Cys-297, is located close to the C terminus and connects two -helices. Here we demonstrate that the formation of these disulfide bonds is well ordered in time. One disulfide bond is formed in the proenzyme and is essential for subsequent autoproteolytic processing to occur. The other disulfide bond is formed only after autocatalytic processing and appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Growth Conditions-- The bacterial strains used are listed in Table I. To allow for replication of pKNG101 derivatives, Escherichia coli strain CC118(pir) was used. Selection for mutant forms of elastase was done with E. coli strain DH5. For isolation of other plasmids, E. coli strain PC2495 was used. Cells of E. coli and P. aeruginosa were grown in LB medium with agitation at 37 °C, unless otherwise indicated. For LB-agar plates, the medium was solidified with 1.5% agar. The antibiotic concentrations used for plasmid maintenance were 25 and 20 µg/ml kanamycin and 100 and 500 µg/ml streptomycin for E. coli and P. aeruginosa, respectively, 100 µg/ml ampicillin for E. coli, and 20 µg/ml piperacillin for P. aeruginosa. The dsbC gene in E. coli strain CE1224 was inactivated by P1 transduction (22), using the dsbC::Km strain SR3515 as a donor, and transductants were selected on LB plates, supplemented with kanamycin. The lasB gene in P. aeruginosa strains PAO25 and PAO7510 was inactivated by using pPB55, which is a derivative of the suicide vector pKNG101 containing a lasB::Km allele, essentially as described (23). The lasB mutants were designated PAN10 and PAN11, respectively.

Plasmids and DNA Manipulations-- The plasmids used are listed in Table II. Plasmid isolations from E. coli, restriction endonuclease digestions, ligations, and agarose gel electrophoresis were performed according to standard procedures (24). The enzymes used were purchased from Amersham Pharmacia Biotech or Fermentas. DNA fragments were isolated and purified from agarose gel using the JetSorb gel extraction kit 600 (Genomed Inc.).

Nucleotide sequences were determined on double-stranded plasmid DNA using the Big Dye terminator cycle sequencing kit and the ABI Prism 310 automated sequencer (PerkinElmer Life Sciences), according to the manufacturer's instructions.

For the insertional inactivation of the lasB gene, plasmid pPB3, encoding pre-elastase without the propeptide, was digested with PstI, and the kanamycin resistance box of plasmid pUC4K was cloned as a PstI fragment in this site. From the plasmid obtained, designated pPB12, the modified lasB gene was isolated as an EcoRI-SphI fragment, which was made blunt with T4 DNA polymerase and cloned in the SmaI site of the suicide vector pKNG101, resulting in plasmid pPB55.

Cys-30 and Cys-57 were simultaneously replaced by alanine residues, using a modified QuickChange^TM site-directed mutagenesis protocol (Stratagene). Plasmid pRB1804 was used as a template, and the DNA was amplified with the mutagenic oligonucleotides 5'CAACGACCGCgcCGAGATGGACG and 5'TTGGTCGGGgcGGCGAAGCGGAAC (Amersham Pharmacia Biotech) by 20 cycles of denaturation at 94 °C for 1 min, primer annealing at 50 °C for 1 min, and DNA synthesis at 68 °C for 2 min. Transformants encoding elastase without the disulfide bond were elected by the altered migration of elastase using SDS-PAGE¹ analysis, and the nucleotide substitutions were confirmed using nucleotide sequence analysis. Thus, plasmid pUC  NSS was obtained.

To substitute the cysteines forming the C-terminal disulfide bond, first, the substitution C297A was made using the 3-primer method of Landt et al. (25) with the following modifications. The "megaprimer" was obtained by amplification of the DNA fragment with the oligonucleotides 5'AGCGGCGCCgcCGGGGTGAT and 5'cTgcAGCAGCCGCCCTCC in 25 cycles of denaturation at 94 °C for 30 s, primer annealing at 47 °C for 1 min, and DNA synthesis at 73 °C for 2 min. The entire elastase gene with the C297A substitution was obtained using pRB1804 as template, the megaprimer, the reverse sequencing primer, and 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 47 °C for 2 min, and DNA synthesis at 73 °C for 2.5 min. The DNA fragment obtained was digested with EcoRI and PstI and cloned in the corresponding sites of pUC18 resulting in plasmid pUCCS. The second cysteine of the C-terminal disulfide bond, C270, was subsequently substituted, using the QuickChange^TM site-directed mutagenesis method using pUCCS as the template and the oligonucleotides 5'GGCGTGACCgcCCCGAGCGC and 5'GCGCTCGGGgcGGTCACGCC. The QuickChange mutagenesis was carried out as described above, except that the annealing temperature in the polymerase chain reaction was 55 °C. Thus, plasmid pUCCSS was obtained.

For expression in P. aeruginosa, the mutant lasB alleles were isolated as EcoRI/PstI fragments from pUCNSS and pUCCSS and cloned in the corresponding sites of pMMB67EH, resulting in plasmids pMMBNSS and pMMBCSS, respectively.

Transformation and Mobilization-- Transformation of E. coli strains and transfer of plasmid DNA to P. aeruginosa by triparental mating, using the conjugative properties of pRK2013, were done as described (13). P. aeruginosa transconjugants were selected on King's medium B agar plates (26), supplemented with 20 µg/ml naladixic acid.

Pulse Labeling and Immunoprecipitations-- Growth of the cultures, the induction of elastase expression from the tac promoter, pulse labeling of the cells, and immunoprecipitations were performed as previously reported (13). Where indicated, oxidation of reduced thiols by air was prevented by incubating the pulse-labeled cells for 20 min at 0 °C with 100 mM iodoacetamide (Sigma), according to standard procedures (27, 28). Subsequently, the proteins were precipitated with trichloroacetic acid. A polyclonal antielastase serum was made using purified mature elastase (Nagase Laboratory) that was injected into a rabbit. The serum obtained was used in a 1000-fold dilution and was a kind gift from A. Lazdunski. The polyclonal antipropeptide serum was a generous gift from and prepared by E. Kessler (15). In short, the propeptide was isolated from the partial purified elastase precursor using SDS-PAGE. Gel slices containing the antigen were homogenized, mixed with complete Freund's adjuvant, and injected into rabbits. The IgG fraction was purified from the blood samples by DEAE-cellulose chromatography. The serum was preadsorbed with a cell extract of P. aeruginosa strain AP103-II and E. coli strain DH5(pUC18). The thus 20-fold diluted serum was used in Western blotting analysis using a 20,000-fold final dilution.

Separation of Intracellular and Extracellular Proteins-- To obtain cell-free extracellular fluids, cells were removed from cultures by centrifugation (6500 × g, 3 min, room temperature), and the supernatant obtained was centrifuged again (13,000 × g, 3 min, room temperature). Proteins were precipitated with 5% trichloroacetic acid and washed with acetone. The precipitated proteins and the cells obtained in the pellet of the first centrifugation were resuspended in sample buffer without dithiothreitol (DTT).

SDS-PAGE, Western Blotting, and Detection of Free Thiols-- Trichloroacetic acid-precipitated or immunoprecipitated proteins were resuspended in sample buffer (29) without -mercaptoethanol. For the reduction of disulfide bonds, DTT was added to a concentration of 20 mM. Protein patterns were analyzed by SDS-PAGE (13) on gels of 9 cm in length (Figs. 2 and 3) or on the Bio-Rad Protean II system (Figs. 1 and 4) followed, where indicated, by autoradiography. Immunodetection by Western blot analysis was performed as described (30). To detect free thiols, proteins were incubated with N-[6-(biotinamido)]hexyl-3'(2'pyridyldithio)propionamide (biotin-HPDP) as described (28). Briefly, proteins trichloroacetic acid-precipitated from culture supernatants were incubated for 10 min at 95 °C in the presence or absence of DTT. Subsequently 20 mM phosphate-buffered saline, 10 mM EDTA, supplemented with 0.8 mM biotin-HPDP (Pierce), was added, and the samples were incubated for 1 h at room temperature. Next the proteins were separated by SDS-PAGE and blotted onto nitrocellulose filters (Schleicher and Schuell, 0.45 µm) using a semidry electroblotting apparatus (2117 Multiphor II, LKB). Streptavidin-horseradish peroxidase was used to detect biotin-labeled proteins. The peroxidase activity was developed with a solution of 4-chloro-1-naphthol (0.5 mg/ml) in 15% methanol, 85% phosphate-buffered saline, and 0.01% H₂O₂.

Enzyme Assay-- The proteolytic activity of elastase in culture supernatants of P. aeruginosa was determined as previously described (31).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Disulfide Bonds in Elastase-- Before studying the formation of disulfide bonds during the biogenesis of elastase, we first analyzed whether their presence can be demonstrated by SDS-PAGE. Frequently polypeptide chains possessing disulfide bonds have a higher electrophoretic mobility than the reduced forms of these polypeptides because of their more compact shape. Indeed when proteins from the supernatant of an overnight culture of the wild-type strain PAO25 were analyzed in the absence of DTT, elastase migrated with a higher electrophoretic mobility (Fig. 1A, lane 1) than in the presence of the reducing agent (Fig. 1A, lane 2). The absence of free cysteines in elastase was further demonstrated by incubating the proteins with biotin-HPDP, which reacts with free thiols. Biotin-HPDP reacted only with elastase after reduction of the disulfide bonds (Fig. 1B). We conclude that the presence or absence of disulfide bonds in mature elastase can indeed be demonstrated by SDS-PAGE.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Disulfide bonds in mature elastase. Cells of P. aeruginosa strain PAO25 were grown overnight at 37 °C, and the supernatant was isolated. Proteins were precipitated with trichloroacetic acid and resuspended in sample buffer without or with DTT, as indicated. Samples were subsequently analyzed by SDS-PAGE (A, samples corresponding to a 180-µl culture) or first labeled with biotin-HPDP, prior to SDS-PAGE and transfer to nitrocellulose filters (B, samples corresponding to a 90-µl culture). To detect the biotin-labeled proteins, the blot was probed with streptavidin-horseradish peroxidase. Mature elastase forms E0 and E2 are indicated at the right, and molecular mass markers (Mw, in kDa) are indicated at the left. On the original gel in A, the distance between the mature elastase forms E0 and E2 is 1.5 mm.

To examine the formation of disulfide bonds in elastase, E. coli strain CE1224 was transformed with the lasB-containing plasmid pML27, and elastase maturation was studied after pulse labeling of the cells. Two forms of the proenzyme were detected, tentatively designated PE₀ and PE₁ (Fig. 2, lane 1). Reduction of the proteins with DTT before SDS-PAGE eliminated the PE₁ form (Fig. 2, lane 2) and resulted in a concomitant increase in the amount of the PE₀ form (Fig. 2, lane 2). This result shows that the bands represent different conformational states of the proenzyme and that the proenzyme form PE₁ contains at least one disulfide bond.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Formation of disulfide bonds in elastase expressed in E. coli. Wild-type and dsb mutant E. coli strains were grown to an A600 of 0.5 at 30 °C. Thirty minutes after the induction of elastase expression, the cells were labeled for 3 min, followed by a chase period of 5 min. To prevent spontaneous oxidation of the free thiol groups by air, cells were incubated with iodoacetamide after the chase. Proteins from total cells were precipitated with trichloroacetic acid and dissolved in Triton buffer, and elastase was immunoprecipitated. Samples corresponding to a 200-µl culture were resuspended in sample buffer with or without DTT, as indicated, and applied to SDS-PAGE and analyzed by autoradiography. To prevent diffusion of DTT to neighboring lanes, empty lanes were used to separate samples with and without DTT. These lanes, including the ones containing nonrelevant samples, were removed from the figure for clarity. Lanes 1 and 2, CE1224(pML27) (wild-type); lanes 3 and 4, CE1442(pML27), (dsbA::Km); lanes 5 and 6, CE1467(pML27), (dsbC::Km). Proelastase forms PE0 and PE1 and mature elastase forms E0, E1, and E2 are indicated. On the original gel, the distances between the mature elastase forms E0 and E1 and E1 and E2 are 1.0 and 1.5 mm, respectively.

Two forms of mature elastase were detected, a faint band tentatively designated E₁ and a major band designated E₂ (Fig. 2, lane 1). When the proteins were exposed to DTT before SDS-PAGE, both the E₁ and the E₂ forms of the mature elastase were converted into another form, designated E₀, which migrated even slower in the gel than E₁ (Fig. 2, lane 2). Hence it appears that three different forms of mature elastase can be discriminated, which differ in the number of disulfide bonds. Form E₀ is the fully reduced state of the protein, whereas E₁ and E₂ contain most likely one and two disulfide bonds, respectively. Because the E₁ form could be detected after expression of elastase in vivo, it is apparently not necessary that both disulfide bonds are formed for autoproteolytic processing to occur (see also below).

Disulfide Bond Formation in Elastase Expressed in P. aeruginosa-- Because the experiments described above were performed in E. coli, the possibility that the slow formation of the second disulfide bond in mature elastase is an artifact of elastase expression in a heterologous host, rather than a genuine step in the biogenesis of elastase, had to be investigated. Therefore, the maturation of elastase was also studied in P. aeruginosa lasB mutant strain PAO-E105 carrying pML27 in pulse-labeling experiments. The various forms of proelastase (PE₀, PE₁) and mature elastase (E₁, E₂), which were previously detected in E. coli, were also detected in P. aeruginosa, but their relative abundance varied from experiment to experiment. Two extreme cases are illustrated in Fig. 3, A and B. In the experiment illustrated in panel A, the majority of the proenzyme and the mature enzyme detected immediately after pulse labeling of the cells was already in the oxidized states PE₁ and E₂, respectively (Fig. 3A, compare lanes 1-5 with 6). In the experiment illustrated in panel B, the proenzyme is in the reduced state PE₀ (Fig. 3B, lane 1), whereas the mature enzyme is slowly converted during the chase from the E₁ form into the E₂ form (Fig. 3B, lanes 1-5). Apparently, as in E. coli, (i) at least one disulfide bond is already formed in the proenzyme, (ii) the second bond is not required for autoproteolytic processing to occur, and (iii) this second bond can be formed in the already processed enzyme. Therefore, we conclude that the shift in electrophoretic mobility of the mature elastase reflects a relevant step in the biogenesis of the enzyme, which is the formation of the second disulfide bond after the autoproteolytic processing of the mature enzyme.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Formation of disulfide bonds in elastase expressed in P. aeruginosa. Cells of P. aeruginosa strain PAO-E105, carrying plasmid pML27 encoding elastase, were grown to an A600 of 0.35 at 30 °C. Thirty minutes after the induction of elastase expression, the cells were labeled for 3 min, followed by varying chase periods as indicated. Proteins from total cells were precipitated with trichloroacetic acid and resuspended in Triton buffer, and elastase was immunoprecipitated. A and B represent two independent experiments. Samples corresponding to a 200-µl culture were resuspended in sample buffer with or without DTT, as indicated, and analyzed by SDS-PAGE and autoradiography. The proelastase forms PE0 and PE1 and the mature elastase forms E0, E1, and E2 are indicated. On the original gel in A, the distance between the mature elastase forms E0 and E2 is 2.5 mm. In B, the distance between the mature forms E1 and E2 is 1.5 mm.

Role of DsbA and DsbC in Disulfide Bond Formation-- Although the formation of disulfide bonds between free thiols can occur by spontaneous oxidation, their formation is generally catalyzed in vivo by the Dsb proteins in the periplasm. To study the putative role of DsbA and DsbC in the formation of the disulfide bonds in elastase, the protease was expressed in dsbA and dsbC mutant derivatives of E. coli strain CE1224, and elastase maturation was studied after pulse labeling of the cells. The total amount of elastase detected in the dsbA mutant after pulse labeling of the cells was strongly reduced when compared with the wild-type strain (Fig. 2, compare lane 4 with 1). Of the two proenzyme forms detected in the wild-type strain, only the form lacking the disulfide bonds (PE₀) was detected in the dsbA mutant (Fig. 2, lane 4). Apparently the formation of the disulfide bond in the proenzyme is normally catalyzed by DsbA. Furthermore, a strong accumulation of the mature elastase form E₁ relative to E₂ was observed (Fig. 2, compare lanes 1 and 4). This result confirms that the formation of the second disulfide bond is not required for processing to occur and indicates that DsbA also catalyzes the formation of the second disulfide bond in the processed enzyme. Furthermore, because the E₀ form of the mature enzyme could not be detected in the dsbA mutant, it again appears that the formation of the first disulfide bond in the proenzyme precedes autocatalytic processing. The conversion of the proenzyme form PE₀ into mature elastase did occur, but at a reduced rate because the ratio between proenzyme and mature elastase was increased (Fig. 2, compare lanes 1 with 4). These data suggest that (i) either the formation of the first disulfide bond is not essential for processing to occur, even though it stimulates the process, or (ii) alternatively, the disulfide bond is formed slowly, either spontaneously or catalyzed by other Dsb proteins, which allows for subsequent rapid processing (see below).

In the dsbC mutant strain CE1467, the two forms of the proenzyme, PE₀ and PE₁, as well as the two forms E₁ and E₂ of the mature enzyme were detected (Fig. 2, lane 5). These forms were also observed in the wild-type strain. Therefore, DsbC does not seem to be essential in the formation of the disulfide bonds in elastase, consistent with its role as a reductase, rather than as an oxidase (32).

Role of Individual Disulfide Bonds in Elastase Maturation-- To determine which of the two disulfide bonds is first formed and whether the formation of this disulfide bond is indeed essential for the processing of the proenzyme, two lasB mutants were isolated (see "Experimental Procedures"). In each of the mutants one pair of cysteines was replaced by alanines. The proteins lacking the N-terminal or the C-terminal bond were expressed from pMMBNSS and pMMBCSS, respectively. Their maturation was analyzed in P. aeruginosa using SDS-PAGE and Western blotting.

Expression of the wild-type elastase from pML27 in the lasB mutant strain PAN10 resulted in the production and secretion of mature elastase (E₂), which was detected both extracellularly (Fig. 4B, lane 2) and, as a consequence of overproduction, intracellularly (Fig. 4A, lane 2). When the gene encoding elastase without the N-terminal cysteines was expressed in the same strain, the mature protein was detected in the supernatant (Fig. 4B, lane 3). As expected from the absence of a disulfide bond, the mutant protein displayed a slightly lower electrophoretic mobility than the wild-type protein. In contrast to the wild-type enzyme, the mutant enzyme did not accumulate intracellularly (Fig. 4A, lane 3). Consistent with the much lower total amount of enzyme detected, this suggests that the N-terminal disulfide bond contributes to the overall stability of the mature enzyme. Secretion of the mutant elastase was normally dependent on the Xcp machinery because the enzyme was not detected in the supernatant of the xcpR mutant strain PAN11 (Fig. 4B, lane 7) where it accumulated intracellularly (Fig. 4A, lane 7). These results demonstrate that the N-terminal disulfide bond is not required for the autoproteolytic processing of the proenzyme and for proper secretion of the mature protein. Using an antiserum directed against the propeptide, two forms of the propeptide, P₁ and P₂, were found to accumulate in the extracellular medium (Fig. 4C, lane 3). These two forms of the propeptide were previously detected in the supernatant of late-log phase cell cultures, when the extracellular proteolytic activity is still low, but disappeared during prolonged incubation when the proteolytic activity increased (6). These results suggest that, although the N-terminal disulfide bond is not essential for processing, it is required for the full proteolytic activity of elastase, which is apparently required for the extracellular degradation of the propeptide.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Expression of mutant forms of elastase in P. aeruginosa. Cells were grown overnight at 37 °C in the presence of 0.5 mM isopropyl-1-thio--D-galactopyranoside. Cells and cell-free culture supernatant were separated, and samples of the cells and supernatant corresponding to 50-µl and 360-µl cultures, respectively, were analyzed by SDS-PAGE prior to transfer to nitrocellulose filters. The blot was probed with antielastase (A, B) or antipropeptide (C) antiserum, respectively. Lane 1, PAN10(pMMB67EH); lane 2, PAN10(pML27); lane 3, PAN10(pMMBNSS); lane 4, PAN10(pMMBCSS); lane 5, PAN11(pMMB67EH); lane 6, PAN11(pML27); lane 7, PAN11(pMMB NSS); lane 8, PAN11(pMMBCSS). Elastase form 1 (E1), elastase form 2 (E2), propeptide form 1 (P1), propeptide form 2 (P2), and proelastase (PE0) are indicated at the right and molecular mass markers (in kDa) are indicated at the left. On the original gel in A and B, the distance between the mature elastase forms E1 and E2 is 1.0 mm. In C, the distance between the propeptide forms P1 and P2 is 1.5 mm.

When the mutant elastase lacking the C-terminal disulfide bond was expressed in the lasB strain PAN10, mature elastase was neither intracellularly (Fig. 4A, lane 4) nor extracellularly (Fig. 4B, lane 4) detected. Instead, the proenzyme (PE₀) was found to accumulate intracellularly (Fig. 4, lane 4). Apparently the C-terminal disulfide bond is formed first, and its formation is required for the processing of the proenzyme.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Extracellular elastase of P. aeruginosa contains two disulfide bonds. In this study, we show that the two disulfide bonds are formed successively. The first disulfide bond, between Cys-270 and Cys-297, is already formed in the periplasmic proenzyme. The formation of this bond is catalyzed by DsbA and is essential for the autoproteolytic processing of the proenzyme (Fig. 4). This conclusion is in agreement with the notion that the mature form E₀ without disulfide bonds was never detected in vivo, not even in a dsbA mutant. However, in the dsbA mutant, some mature enzyme containing one disulfide bond was formed (Fig. 2, lane 4), which is probably the result of spontaneous oxidation or catalysis by other Dsb proteins. The tertiary structure of elastase is similar to that of Bacillus cereus thermolysin (21). However, thermolysin does not contain disulfide bonds, and such bonds are apparently not required for the autocatalytic processing of this enzyme.

The formation of the second, N-terminal disulfide bond, which is formed after cleavage of the propeptide in the processed enzyme, is almost completely inhibited in the dsbA mutant of E. coli, which suggests that DsbA mediates the formation of this bond. Importantly, it has been demonstrated that DsbA binds preferentially to unfolded proteins (33). Furthermore, Eppens et al. (28) showed that the DsbA-catalyzed formation of disulfide bonds in a mutant PhoE protein is extremely rapid and precedes the formation of the (trypsin-resistant) folded conformation. Our observation that DsbA mediates the formation of a disulfide bond in active and thus folded elastase is somewhat unexpected and suggests that DsbA not only mediates the formation of disulfide bonds in unfolded but also in folded polypeptides.

Recently we showed that the propeptide and the mature elastase are both secreted and that the propeptide is degraded extracellularly (6). When the proteolytic activity of extracellular elastase and other Ca²⁺-dependent proteases was prohibited by growth of the cells in a medium depleted of Ca²⁺ ions, the propeptide accumulated extracellularly. Now we found that the propeptide also accumulated extracellularly, when elastase lacking the N-terminal disulfide bond was produced. The mutations did not interfere with the autoproteolytic processing and with the secretion of the mature enzyme but they prohibited the degradation of the propeptide. Consistently the proteolytic activity of the mutant enzyme in the extracellular medium was found to be severely reduced (data not shown). These results suggest that the N-terminal disulfide bond, besides its role in stabilizing the mature enzyme, is required for full proteolytic activity. In that case, the results would underscore the supposition that the propeptide is degraded by elastase itself. Alternatively the propeptide remains associated to the mutant enzyme and thereby inhibits the proteolytic activity. In that case, the formation of the N-terminal disulfide bond would be essential for the release of the propeptide to occur. Interestingly when the wild-type elastase was co-expressed with the mutant enzyme, it was not capable of degrading the propeptide of the mutant form (data not shown). Apparently each enzyme molecule can only degrade its cognate propeptide. A possible explanation for this result is that if the propeptide remains associated with the mature enzyme, it is protected against proteolysis by the wild-type enzyme.

In the past few years, much information concerning the order of events during the biogenesis of elastase has accumulated. To include the data presented in this paper, we have adapted the model for elastase secretion presented by Kessler and Safrin (15). The new model is schematically shown in Fig. 5. First, elastase is synthesized in the cytoplasm as a preproprotein. The pre- part is the signal peptide, which mediates translocation across the inner membrane via the Sec machinery (15). In the periplasm, the propeptide mediates the folding of the mature domain (12, 13) into its active conformation. Here we demonstrate that first a disulfide bond is formed between Cys-270 and Cys-297 and that the formation of this bond is catalyzed by DsbA. This bond is rapidly formed in the proenzyme, probably before the proenzyme is fully folded and before it is proteolytically processed. The formation of this bond is essential to allow for the subsequent autocatalytic processing of the proenzyme. After processing, which is a prerequisite for secretion (34), the propeptide remains noncovalently associated with the mature enzyme (15) and thereby inhibits the premature activation of the proteolytic activity of elastase in the periplasm (17). In addition, we showed that the second disulfide bond, between Cys-30 and Cys-57, is formed after processing in the mature enzyme and that its formation is also catalyzed by DsbA. The entire propeptide-enzyme complex is translocated across the outer membrane (6, 18). Dissociation of the propeptide-enzyme complex is a well coordinated process. The autoproteolytic processing of proelastase per se is not the trigger for the dissociation of the propeptide-enzyme complex, but interaction with a host-specific factor, possibly the Xcp secretion machinery, is required to induce this event (19). After the dissociation of the propeptide-enzyme complex, which occurs during or shortly after translocation across the outer membrane, the propeptide is degraded by an extracellular protease (6). This process requires a fully active mature elastase.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Model for the maturation and transport of elastase. The sequential order of events, from the synthesis of the preproenzyme in the cytoplasm up to the activation of elastase in the extracellular medium, is described in the text. Proteins are schematically drawn. The Sec machinery (Sec) and the Xcp machinery (Xcp) are drawn as single units, for clarity. Also the Xcp outer membrane translocation pore is indicated by the XcpQ protein. Outer membrane (OM), inner membrane (IM), DsbA (DsbA), disulfide bonds (s-s), signal peptide (Sp), propeptide (P, dark domain), and elastase (E, light domain) are indicated.

	ACKNOWLEDGEMENTS

We thank A. Lazdunski for providing antielastase antiserum and P. aeruginosa strain AP103-II and S. Raina for providing E. coli strain SR3515.

	FOOTNOTES

^* This research was supported by the Netherlands Foundation for Chemical Research (SON) and by the Life Sciences Foundation (SLW), which are subsidized by the Netherlands Organization for Scientific Research (NWO) and by European Community E.U. Grant bio4-CT960119.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

^§ Present address: Dept. of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

^¶ Present address: Dept. of Medical Microbiology, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

To whom correspondence should be addressed: Dept. of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-2532999; Fax: 31-30-2513655; E-mail: j.p.m. tommassen@bio.uu.nl.

Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M007122200

	ABBREVIATIONS

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; HPDP, N-[6-(biotinamido)]hexyl-3'(2'- pyridyldithio)propionamide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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