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J Biol Chem, Vol. 273, Issue 46, 30225-30231, November 13, 1998
From the Pseudomonas aeruginosa elastase and
the LasA protease are synthesized as preproenzymes with long
amino-terminal propeptides. The elastase propeptide is cleaved
autocatalytically in the periplasm to form a transient, inactive
elastase-propeptide complex. In contrast, the processing of proLasA
does not involve autoproteolysis. In this study, we analyzed short-term
P. aeruginosa cultures under conditions that minimize
proteolysis and found that an elastase-propeptide complex is secreted,
and then the propeptide is degraded extracellularly, apparently by
elastase itself. LasA protease, on the other hand, was found to be
secreted in its unprocessed 42-kDa proenzyme form. The processing of
proLasA occurred extracellularly, and it involved the transient
appearance of a 28-kDa intermediate and the respective 14-kDa LasA
propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also
proceeded via the 28-kDa intermediate, but the rate of processing was
greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was
blocked. Purified secreted proteases of P. aeruginosa
(i.e. elastase, the lysine-specific protease, and alkaline
proteinase) converted proLasA to the active enzyme. Processing by
elastase and the lysine-specific enzyme, but not by alkaline
proteinase, proceeded via the 28-kDa intermediate, and both were far
more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted
with their propeptides, which are then degraded by secreted proteases
of P. aeruginosa. In addition to their other functions, the
propeptides may play a role in targeting their respective enzymes
across the outer membrane.
Pseudomonas aeruginosa, a Gram-negative opportunistic
pathogen, secretes several proteases into its environment that are
implied in the pathogenicity of the organism. This group of enzymes
includes at least four endopeptidases, elastase (also termed
pseudolysin; Refs. 1-3), alkaline proteinase (1), LasA protease
(staphylolysin; Refs. 3 and 4), and a lysine-specific endopeptidase
that cleaves the peptide bonds following lysine residues in peptides and proteins (5). Elastase and LasA protease can degrade elastin (1, 6,
7), and LasA protease also has high staphylolytic activity that results
from cleavages of the pentaglycine cross-linkages in the cell wall
peptidoglycan (3, 4, 8). LasA protease action on proteins, including
elastin, is limited and directed mainly to Gly3 and
Gly2-Ala sequences that are uncommon in most proteins.
Elastase, on the other hand, is a potent protease that cleaves proteins
at multiple sites and can degrade many host proteins in addition to
elastin (1, 2). Although alkaline proteinase has a broad cleavage
specificity, it is not as potent as elastase and has no elastolytic activity.
Elastase is a 33-kDa zinc metalloendopeptidase that is closely related
to thermolysin (2, 3). Encoded by lasB, elastase is
synthesized as a preproenzyme (53.4 kDa) with a classical signal peptide and a covalently linked 18-kDa amino-terminal propeptide (9-11). The 2.4-kDa signal sequence is removed upon passage through the inner membrane into the periplasm, where the propeptide is rapidly
cleaved off by autoproteolysis (12). This step is apparently necessary
for elastase secretion because an active site mutation blocks the
secretion of elastase as well as enzymatic activity and processing
(13). When lasB is expressed in Escherichia coli, autocatalytic cleavage of the propeptide occurs independently of any
other P. aeruginosa gene product(s) (12). The elastase propeptide acts as an inhibitor of the enzyme (14, 15). After autoprocessing, it remains noncovalently associated with the mature domain to form an inactive propeptide-elastase complex, and it is in
this form that elastase is found within the periplasm of P. aeruginosa (10, 14, 15). The propeptide also functions as an
intramolecular chaperone required for correct folding and secretion
competence (16, 17). Elastase translocation through the outer membrane
is mediated by a complex extracellular protein export apparatus
(Xcp) of at least 12 genes that bears significant homology
to the pullulanase (type II; general secretory pathway) export
apparatus of Klebsiella oxytoca (18). Although the
propeptide is required for elastase secretion, the mechanisms involved
and the fate of the propeptide remain unknown.
LasA protease is a 20-kDa zinc metalloendopeptidase belonging to the
Here we show that in P. aeruginosa, both LasA protease and
elastase are secreted with their propeptides. However, whereas LasA
protease is exported in its unprocessed proenzyme form, elastase seems
to be secreted as a noncovalent complex with its propeptide. The
propeptides of both enzymes are degraded extracellularly by the action
of elastase and other secreted proteases, and the processing of the
LasA propeptide proceeds via a distinct 28-kDa intermediate. The
corresponding 14-kDa propeptide fragment is also detected.
Bacterial Strains and Culture Conditions--
E. coli
strains JM109(pJKG107) with lasA under the inducible
trc promoter (20) and JM109::DE3(pETPRO) with the
lasA propeptide coding sequence fused to a 6× His tag and
cloned into the NotI site of pET28b (Invitrogen) were grown
in L broth containing ampicillin (100 µg/ml) or kanamycin (30 µg/ml), respectively. Prototrophic P. aeruginosa strain
FRD2 and its lasB Preparation of P. aeruginosa Cell Extracts and Culture
Supernatants--
Culture supernatants for assays of proteolytic
activity were prepared by centrifugation (8,000 × g
for 20 min) of the cell suspensions at 4 °C. To prepare samples for
SDS-PAGE1 and immunoblotting
analyses, P. aeruginosa cells were harvested by
centrifugation (Beckman microfuge; 1 min) at room temperature, and the
cells were suspended in 10% TCA. The supernatants were recentrifuged
for 4 min to remove the remaining bacteria and supplemented with bovine
serum albumin (100 µg), and the proteins in the supernatants were
immediately precipitated with 10% TCA. After standing overnight at
room temperature, the TCA precipitates were pelleted, washed with
acetone, air-dried, and dissolved in SDS sample buffer (1:10 of
original volume; Ref. 21). For immunoblotting under nondenaturing conditions, samples of the cell cultures were centrifuged (Beckman microfuge; 5 min) at 4 °C, and 0.5-ml aliquots of the cell-free supernatants were immediately transferred into chilled tubes containing 50 µl of TLCK (50 mM) and 1,10-orthophenanthroline (20 mM) in water. After standing on ice for additional 20 min,
these samples were frozen and stored at Production and in Vitro Processing of proLasA--
Production of
proLasA was induced in E. coli JM109(pJKG107) with 1 mM isopropyl-1-thio- Purification of LasA Protease and Its Propeptide--
LasA
protease was purified from the culture supernatant of P. aeruginosa strain FRD2 by DEAE-cellulose chromatography (8), followed by Cm-cellulose chromatography. The Cm-cellulose column (2.2 × 23 cm) was equilibrated with 0.02 M Tris-HCl
and 0.5 mM CaCl2, pH 8.0, and eluted with a
linear gradient of NaCl (400 ml; 0-0.6 M). The enzyme
emerged from the column at ~0.4 M NaCl and, as judged by
SDS-PAGE and silver staining (data not shown), was homogenous.
Fractions containing staphylolytic activity (8) were concentrated by
ultrafiltration (Diaflo YM-10 membrane), dialyzed against 0.05 M Tris-HCl, pH 8, and stored in aliquots at
Antibodies--
Antibodies to LasA protease were raised in
rabbits (14), purified from the IgG fraction by adsorption to LasA
protease immobilized on nitrocellulose blots (23), and used at
dilutions of 1:500 to 1:1,000. Antibodies to the propeptide of LasA
protease were raised in rabbits as described previously (20). These
antibodies were isolated from the immune serum (diluted 1:100 in 30 ml
of Tris-buffered saline plus 0.05% Tween 20 containing 1% bovine serum albumin) by adsorption to proLasA from cell extracts of E. coli JM109(pJK107) immobilized on nitrocellulose blots (24, 25).
The antibody solution was concentrated with sucrose (20), dialyzed into
Tris-buffered saline plus 0.05% Tween 20, and used in immunoblots at a
1:500 dilution. Previously described rabbit antibodies to the elastase
propeptide (10) were purified from the IgG fraction by adsorption to
nitrocellulose-immobilized propeptide (23). The propeptide required for
this was derived from P. aeruginosa strain
FRD740(pSDM3.7tac) (16). The cells were washed with saline and broken
by sonication in 0.02 M Tris-HCl and 0.5 mM
CaCl2, pH 7.5, containing the protease inhibitors leupeptin
(1 µg/ml), benzamidine (10 mM), and
4-(2-aminoethyl)-benzenesulfonyl fluoride (1 mM; also named
Pefabloc; Merck). The soluble cell fraction was prepared by
ultracentrifugation (100,000 × g for 1 h at
4 °C), and a sample of this fraction containing approximately 15 µg of propeptide was electroblotted to nitrocellulose. The region corresponding to the propeptide was identified by staining with Ponceau
S and used after excision for immunoadsorption (23). The
elastase-propeptide antibody was used at a 1:1,000 or 1:100 dilution
for immunoblots of SDS or nondenaturing gels, respectively. Immunoaffinity-purified antibodies to denatured elastase (10) were used
at a dilution of 1:10,000.
Polyacrylamide Gel Electrophoresis and
Immunoblotting--
SDS-PAGE was performed according to Laemmli (21)
with 4% stacking gels and 12% separating gels. Electrophoresis under
nondenaturing conditions was performed according to the method of Davis
(26), except that the gel and electrode buffers contained 5 and 1 mM 1,10-phenanthroline, respectively. The electroblotting
of proteins from SDS gels to nitrocellulose was performed in
ethanolamine buffer, pH 9.4 (27). The transfer of proteins from
nondenaturing gels was performed in Tris (7.5 mM)-borate
(1.2 mM) buffer, pH 8.9, containing 1,10-phenanthroline (1 mM). These blots were treated with 3% TCA for 30 min,
washed with Tris-buffered saline, and then incubated for 30 min at
50 °C in 62.5 mM Tris-HCl, pH 6.7, containing 2% SDS
and 100 mM 2-mercaptoethanol to denature the proteins. All
blots were blocked with 3% skim milk in Tris-buffered saline, and
after incubation with a specific antibody, immunoreactive bands were
detected with peroxidase-conjugated goat anti-rabbit IgG (Sigma) and
enhanced chemiluminescence.
Secretion and Extracellular Processing of proLasA--
Our
previous studies on the processing and secretion of LasA protease
relied on analyses of overnight cultures of P. aeruginosa. The results of these studies indicated that the proteolytic processing of proLasA is not autocatalytic and may involve other secreted proteases of P. aeruginosa (20). The processing protease,
its cellular location, and the stage during secretion at which
processing occurs remained unknown. To better understand the mechanisms
underlying the maturation and secretion of the LasA protease, in the
present study, we defined conditions that allow the identification of short-lived secreted protein species. Late-log P. aeruginosa
cells were suspended in fresh medium, samples were removed at short intervals after medium replacement, and TCA was added to both the cell
and medium fractions to prevent proteolysis. Immunoblots with
antibodies to LasA protease revealed that at any of the time points
examined, the cells contained only proLasA (Fig.
1A). Although the amount of
cell-associated proLasA decreased during the 2-h period of the
experiment, this was not accompanied by the appearance of smaller
LasA-related proteins, suggesting that no processing of proLasA had
occurred within the cells. Consistent with this, medium samples taken
at early time points contained proLasA as the only LasA-related protein
(Fig. 1, B and C, 0 and 10 min). Extracellular
processing was evident within 20 min and proceeded in a
time-dependent manner, with just a trace of proLasA
remaining after 2 h (120 min). At 20 min, small amounts of
mature-size (20 kDa) LasA protease and a 28-kDa processing intermediate
were detected in the medium, and their levels increased with time, in
parallel to the disappearance of proLasA (40-120 min). The 28-kDa
processing intermediate apparently resulted from cleavage(s) within a
region in the propeptide that is sensitive to proteolysis (rather than multiple random cleavages) because a 14-kDa propeptide fragment recognized by antibodies to the LasA propeptide but not by anti-LasA protease antibodies was also detected (Fig. 1C, 20-120
min). ProLasA and the 28-kDa processing intermediate were visualized
with antibodies to both mature LasA protease and the LasA propeptide
(compare B and C in Fig. 1), and immunoblots of
cell extracts with the LasA propeptide antibody (data not shown)
yielded a pattern indistinguishable from that obtained with the LasA
protease antibody (Fig. 1A), detecting proLasA as the only
cell-associated propeptide-related band. Collectively, these results
indicate that proLasA is not processed in the cell. Instead, proLasA is
exported in its unprocessed form, and the propeptide is degraded
extracellularly in a stepwise manner by the action of other secreted
proteases of P. aeruginosa. Secretion of proLasA appears to
occur rapidly after its formation because, at any time, the level of
cell-associated proLasA was far smaller than that of the secreted
precursor and its extracellular processing products together (compare
A and B in Fig. 1).
Proteases Involved in Extracellular Processing of
proLasA--
Elastase is the prominent and most potent protease
secreted by most P. aeruginosa strains. To examine whether
elastase may play a role in proLasA processing and to further establish
the extracellular location of this reaction, we compared proLasA
secretion and processing in strains FRD2 (wild type) and FRD740
(lasB
To substantiate the involvement of the lysine-specific endopeptidase in
the physiological processing of proLasA, we examined whether TLCK, a
specific inhibitor of this enzyme (5, 7), can block proLasA processing
in cultures of P. aeruginosa strain FRD740
(lasB Elastase Is Secreted with Its Propeptide--
When P. aeruginosa is grown under most conditions, little or no
proelastase is found in the cells, nor is it detectable in the
supernatants (10, 13). Instead, proelastase is rapidly processed in the
periplasm to form an inactive elastase-propeptide complex (10, 15), and
this seems to be required for the efficient secretion of elastase (13).
Previously, we speculated that the propeptide is degraded in the
periplasm (10). The dependence of elastase secretion on the propeptide,
which was recognized in subsequent studies (16, 17), led us to examine
the possibility that elastase is exported along with the cleaved
propeptide. As in the case of LasA protease, elastase secretion was
followed by analyzing samples from short-term cultures of late-log
phase P. aeruginosa cells suspended in fresh medium. To
detect an elastase-propeptide complex, which requires nondenaturing
conditions, the culture supernatants were not treated with TCA, and the
proteolysis of unstable secreted proteins was minimized by the addition
of protease inhibitors (see "Experimental Procedures"). SDS-PAGE
and immunoblotting revealed that supernatant samples removed during the
first 40 min contained elastase (Fig. 5,
top panel) as well as significant amounts of the 18-kDa
propeptide (Fig. 5, bottom panel). Also, a
propeptide-related protein of about 15 kDa, which is likely a
degradation product of the propeptide, was observed. The intensity of
the propeptide band remained approximately the same between 0 and 40 min and decreased rapidly between 40 and 60 min; at 2 h, the
propeptide was no longer detectable. The rapid disappearance of the
propeptide at the later times correlated well with the accumulation of
elastase in the medium (Fig. 5, top panel), suggesting that
elastase was involved in the degradation of its own propeptide.
Immunoblotting of the culture supernatants under nondissociative
conditions revealed that the samples removed during the first 20 min
contained an elastase-related protein band designated E/P that migrated
more slowly than elastase. By 20 min, two additional elastase-related
bands with an intermediate mobility between that of E/P and elastase
were evident, but no free elastase was detectable (Fig.
6, left panel). E/P was
highlighted by antibodies to both elastase and its propeptide (Fig. 6,
left and right panels, respectively) and migrated
identically with the periplasmic elastase-propeptide complex,
suggesting that it represents a noncovalent elastase-propeptide complex. Another band with mobility between that of E/P and elastase and identified in the periplasmic preparation of E/P was visualized with both antibodies and may represent an E/P complex containing partially degraded propeptide. Free elastase appeared by 40 min, and
its level increased further by 60 min, but the elastase-propeptide complex and the band with intermediate mobility were detectable throughout the experiment. Collectively, the above mentioned results indicate that elastase is secreted as a noncovalent complex with its
propeptide, and then the propeptide is degraded extracellularly, most
probably by elastase itself.
Our earlier studies on elastase processing and secretion suggested
that the elastase propeptide might be degraded in the periplasm (10).
The dependence of elastase secretion on its propeptide, which was
subsequently established by us (16) and others (17), raised the
possibility that the propeptide may provide information required for
passage through the outer membrane and thus may be secreted along with
its enzyme. The propeptide of the Secretion of the elastase-propeptide complex, documented here for the
first time, is consistent with our previous findings that indicate that
autoprocessing within the periplasm is essential for efficient elastase
secretion (3, 10). However, an alternative secretion pathway for
elastase involving the secretion of unprocessed proelastase cannot be
excluded. Extracellular proelastase can be observed when autoprocessing
is prevented by site-directed mutagenesis of the elastase active
site,2 or by growing the
bacteria in media depleted of Zn and Ca (30). However, the secretion of
proelastase under such conditions appears to be inefficient. The export
of unprocessed proelastase probably represents a secondary pathway for
elastase secretion, with the primary pathway involving export of the
inactive elastase-propeptide complex.
The demonstration of proLasA as the first LasA-related protein to
appear in the medium and the absence of proLasA-processing intermediates in the cells provide strong evidence that LasA protease is secreted as an unprocessed proenzyme. In contrast to elastase, this
seems to be the primary and perhaps the only pathway for LasA protease
secretion. In agreement with our previous findings (20), the processing
of proLasA does not involve autoproteolysis. It occurs in the
extracellular environment and involves secreted proteases other than
LasA protease itself, in particular, elastase and the lysine-specific
endopeptidase. Processing seems to occur in two steps. At first,
cleavage(s) by either elastase or the lysine-specific endopeptidase
takes place at a site approximately 8 kDa upstream of the promature
junction. This produces a 28-kDa processing intermediate (Figs. 1-4),
and a 14-kDa propeptide fragment is released (Fig. 1). Based on the
cleavage specificity of the proteases involved and the size of the
resulting products, we presume that the lysine-specific endopeptidase
may cleave the bond on the carboxyl side of Lys168
(numbered as in preproLasA; Ref. 20), whereas elastase may cleave a
peptide bond involving one of the few alanines surrounding this lysine
residue. The propeptide fragment and the carboxyl-terminal portion of
the LasA propeptide that remains attached to the mature domain are then
degraded by multiple cleavages, most likely by the combined action of
elastase and the lysine-specific protease. Alkaline proteinase can lead
to LasA protease maturation (Fig. 3), although far less effectively
than elastase and the post-lysine cleaving enzyme, and this may be
important in strains lacking these proteases.
Despite this processing by multiple proteases, the amino terminus of
mature LasA protease is the same. This suggests that the amino-terminal
portion of mature LasA protease has a structure resistant to
proteolysis. However, another mechanism is required to ensure the
complete removal of the short propeptide sequence(s) remaining after
cleavage by any of the three endopeptidases. Conceivably, this may be
accomplished by an aminopeptidase, and we have recently discovered such
an enzyme in the culture media of P. aeruginosa.3 The two
proline residues at positions 2 and 3 of the mature LasA protease may
serve to stop the presumed sequential degradation of the extra
propeptide sequences by the aminopeptidase at the exact promature
junction, because peptide bonds involving the imino group of proline
are resistant to most proteases. This may be an important mechanism in
the absence of propeptide-controlled autoprocessing. The finding that
the first cleavage(s) within the propeptide occurs at a specific site
and generates relatively stable products suggests that the LasA
propeptide may contain two functionally distinct domains. Each domain
may have a considerable degree of tertiary structure, and the two
domains may be connected to each other by a linker in which the
proteolytic processing of the LasA propeptide is initiated.
Conceivably, one of these domains may have folding and targeting
functions, whereas the other may function as an inhibitor of LasA
protease. Based on the sequence homology with proteins of known
functions, a similar domain organization has been predicted for the
propeptide of Elastase and LasA protease are representatives of many prokaryotic and
eukaryotic secreted proteases that are synthesized with long
amino-terminal propeptides. There seems to be a close relationship
between folding, propeptide autoprocessing, and secretion, and this has
been established for elastase (13, 16, 17) as well as other secreted
bacterial proteases bearing amino-terminal propeptides such as
subtilisin E from Bacillus subtilis (32) and After submission of this manuscript, a
publication by Braun et al. (Braun, P., DeGroot, A., Bitter,
W., and Tommassen, J. (1998) J. Bacteriol. 180, 3467-3469) documented the presence of the elastase propeptide and
unprocessed proLasA protease in culture media of P. aeruginosa.
*
This work was supported in part by Cystic Fibrosis Grant
G800 (to E. K.), National Institute of Allergy and Infectious Diseases Public Health Service Grant AI-26187 (to D. E. O.), and Veterans Administration Medical Research Funds (to D. E. O.).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.
§
To whom correspondence should be addressed. Tel.: 972-3-5350392;
Fax: 972-3-5351577; E-mail: ekessler{at}post.tau.ac.il.
The abbreviations used are:
PAGE, polyacrylamide
gel electrophoresis; TCA, trichloroacetic acid; TLCK, tosyl-lysyl-chloromethylketone; E/P, elastase-propeptide complex.
2
J. L. Johnston, K. S. McIver, and D. E.
Ohman, unpublished observations.
3
R. Cahan and E. Kessler, manuscript in preparation.
Elastase and the LasA Protease of Pseudomonas
aeruginosa Are Secreted with Their Propeptides*
§,,
Maurice and Gabriela Goldschleger Eye
Research Institute, Tel-Aviv University Sackler Faculty of Medicine,
Sheba Medical Center, Tel-Hashomer 52621, Israel and the
¶ Department of Microbiology and Immunology, University of
Tennessee and Veterans Administration Medical Center, Memphis,
Tennessee 38163
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-lytic endopeptidase family of proteases (3, 4). Like the -lytic
endopeptidase of Achromobacter lyticus (4), LasA protease is
synthesized as a preproenzyme with an unusually long (22 kDa)
amino-terminal propeptide, which is larger than the mature domain (19,
20). LasA protease seems to be exported via the Xcp system, but, as
compared with elastase, little is known about its maturation and
secretion. When expressed in E. coli, proLasA does not
undergo autoprocessing and shows little or no enzymatic activity (19,
20). Substitution of His120, a putative active site residue
of LasA protease (3, 4), does not prevent the processing of proLasA in
P. aeruginosa, and an enzymatically inactive 20-kDa LasA
protein is found in the culture supernatant (20). Although this
suggests that proLasA processing and secretion in P. aeruginosa may proceed by mechanisms that do not involve
autoproteolysis, no specific proLasA-processing protease has yet been
identified, and the processing site is unknown.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
derivative, FRD740 (16), were grown in
tryptic soy broth without dextrose (Difco; Refs. 8 and 10). In some
experiments, when absorbance at 660 nm of the bacterial cell suspension
reached 2 OD units, the cells were harvested by centrifugation
(6,000 × g for 20 min at 4 °C) and resuspended in
the original volume of fresh medium, and growth was continued for
different time intervals, as indicated. P. aeruginosa strain
FRD740(pSDM3.7) with an amber mutation at the propeptide-mature junction of lasB (16) was grown in tryptic soy broth without dextrose plus 100 µg/ml tetracycline.
70 °C until use.
-D-galactopyranoside
(Sigma; Ref. 20). Cells from 100-ml cultures were harvested by
centrifugation (8,000 × g for 20 min), suspended in 5 ml of 20 mM Tris-HCl, pH 7.5, and broken by sonication.
Cell debris was removed by mild centrifugation (6,000 × g for 20 min at 4 °C), and the soluble cell fraction was
then obtained by ultracentrifugation (100,000 × g for
1 h at 4 °C). Processing solutions (50 µl of 20 mM Tris-HCl and 0.25 mM CaCl2, pH
7.5) contained 50 µg of protein from the E. coli cell
extract and 10 or 50 ng of purified elastase (22), alkaline proteinase
(Nagase), or the lysine-specific protease of P. aeruginosa.
The reaction solutions were incubated at 37 °C for 1 h, and the
reaction was terminated by heating in SDS sample buffer (21). The
lysine-specific endopeptidase required for this experiment was purified
from P. aeruginosa strain FRD2128 by DEAE-cellulose
chromatography as described previously (8), except that the column was
equilibrated and eluted with 0.02 M Tris-HCl, pH 8, instead
of pH 7.5. The enzyme preparation thus obtained was homogenous by
SDS-PAGE and silver staining.
70 °C. The propeptide of LasA protease was expressed in E. coli strain JM109::DE3(pETPRO) and purified from the
soluble fraction of these cells by chromatography on nickel-agarose (Invitrogen).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Secretion and extracellular processing of
proLasA. Late-log cells of P. aeruginosa strain FRD2
were suspended in fresh medium and incubated for 10 min at 37 °C for
reequilibration, and aliquots removed at the time intervals thereafter
(top) were concentrated with TCA and analyzed by SDS-PAGE
and immunoblotting with antibodies to LasA protease (A and
B, for whole cell extracts and supernatants, respectively)
and its propeptide (C, supernatants). Proteins in 10-fold
concentrated samples corresponding to 250 µl of the original cell
culture were applied on each lane, except for lane LasA,
which was loaded with 40 ng of purified LasA protease. Left,
molecular masses in kDa.
). Cells were grown continuously (i.e.
without medium replacement), and aliquots were removed at 30-min
intervals from the late logarithmic phase into the early stationary
phase (3-7 h). Cell and media samples were both analyzed for
LasA-related proteins by immunoblotting, and the level of proteolytic
activity in the supernatants was determined using azocasein as the
substrate. The rate of growth of both strains was also compared and
found to be practically the same (Fig.
2A). The cell fractions of
both strains contained comparable amounts of proLasA, and no smaller
LasA-related proteins were detected in extracts from either strain
(Fig. 2, B and C, compare the top
panels), indicating that proLasA production and secretion in both
strains were similar. However, the rate of proLasA processing in the
culture supernatants of strain FRD740 was reduced as compared with that
found for strain FRD2 (Fig. 2, B and C, compare
bottom panels). During the 3-7-h period examined, virtually no proLasA was detectable in the supernatants of strain FRD2, suggesting that processing in the supernatants of this strain occurred
rapidly after secretion. Culture supernatants of strain FRD740, on the
other hand, contained significant amounts of unprocessed proLasA, which
decreased gradually from 6 h onward, in parallel to the limited
increase in proteolytic activity in the medium (see Fig.
2A). In both strains, processing proceeded via the 28-kDa intermediate. However, this processing intermediate accumulated in the
growth medium of strain FRD740 but not in that of strain FRD2.
Accordingly, the levels of fully processed LasA protease in the medium
of strain FRD2 exceeded those in the medium of strain FRD740 (Fig. 2,
B and C, bottom panels). A correlation
was evident between the rate of extracellular proLasA processing and
the level of proteolytic activity in the medium, which was 6-8-fold
higher at any time in supernatants of strain FRD2 than in those of
strain FRD740 (Fig. 2A). These results reinforced the
extracellular location of proLasA processing and indicated a role for
elastase in this process. However, the demonstration that proLasA can
be processed in the growth medium of strain FRD740 suggested that other
secreted proteases of P. aeruginosa in addition to elastase
may be involved. To investigate this, we examined which of the two best
candidates, alkaline proteinase and the lysine-specific endopeptidase,
could process proLasA in vitro in a manner similar to that
of elastase. Samples of an E. coli extract containing
recombinant (inactive) proLasA were incubated with equal amounts of
purified elastase, alkaline proteinase, or the lysine-specific
endopeptidase, and then proLasA processing was evaluated by
immunoblotting. Fig. 3 shows that each of
the three endopeptidases can convert proLasA to the 20-kDa mature-size
LasA protease. However, whereas both the lysine-specific endopeptidase
and elastase were highly and equally effective in proLasA processing,
alkaline proteinase was relatively inefficient in this regard (compare
lanes 2-4 for digests with 10 ng of protease or lanes
5-7 for digests with 50 ng of protease). Furthermore, whereas
processing by elastase and the lysine-specific endopeptidase proceeded
via the 28-kDa intermediate, no such intermediate was observed with
alkaline proteinase (lanes 3 and 6). Apparently,
alkaline proteinase cleaved the LasA propeptide randomly, generating
multiple fragments that escaped from the gel and thus were not
detectable. Elastase and the lysine-specific protease, on the other
hand, appeared to cleave first a peptide bond(s) within the same region
of the propeptide and generate the 28-kDa intermediate, and then the
remaining carboxyl-terminal portion of the propeptide was degraded.
Complete conversion of proLasA to the 20-kDa LasA protein by any of
these proteases (for alkaline proteinase, this was achieved upon
extended incubation with 50 ng of enzyme) was accompanied by a
comparable increase in staphylolytic activity (data not shown). From
the above mentioned results, it appears that the lysine-specific
endopeptidase, in addition to elastase, may play a role in the
extracellular processing of proLasA in P. aeruginosa,
although alkaline proteinase may complement their action.
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Fig. 2.
Extracellular processing of proLasA in
P. aeruginosa strains FRD2 and FRD740: correlation with
proteolytic activity and role of elastase. Cells were grown at
37 °C, and samples taken at 30-min intervals between 3 and 7 h
(OD at 660 nm, 0.8-2.4) were analyzed for both, extracellular
proteolytic activity and proteolytic processing of proLasA.
A, growth and proteolytic activity in the media of strains
FRD2 and FRD740. B and C, immunoblots with
antibodies to LasA protease of cell extracts (top) and
supernatants (bottom) of strains FRD2 and FRD740,
respectively. Proteins in 10-fold concentrated samples corresponding to
250 µl of the original bacterial cultures were applied on all lanes.
Top, time (in hours) at which each sample was taken.
Left, molecular masses in kDa.
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Fig. 3.
Processing of recombinant proLasA by secreted
proteases of P. aeruginosa. The soluble cell fraction
(50 µg of protein) of E. coli JM109(pKJ107) expressing the
lasA gene from a plasmid was incubated at 37 °C for
1 h with 10 and 50 ng of purified elastase (lanes 2 and
5), alkaline proteinase (lanes 3 and
6), or the lysine-specific endopeptidase (lanes 4 and 7), and the incubation solutions were analyzed by
immunoblotting with antibodies to LasA protease. Lane 1, E. coli cell fraction alone (control). Left, molecular
masses in kDa.
). Late-log cells were suspended in fresh medium and
incubated for an additional hour to allow the initial accumulation of
proLasA in the medium. The suspension was then divided into two equal
portions; TLCK was added to one of these, whereas the other was
supplemented with the same volume of medium, serving as a control. Both
suspensions were incubated for an additional 90 min, and samples of the
supernatants removed at 30-min intervals were then analyzed for LasA
protease-related proteins by immunoblotting. Fig.
4 shows that at 60 min, just before the
addition of TLCK, a small fraction of the secreted proLasA was already
processed to mature-size LasA protease, and the 28-kDa intermediate was also detectable. In the control culture that was incubated without the
addition of TLCK, proLasA processing progressed with time. At 150 min,
much of the proLasA was converted to the 28-kDa intermediate, and
significant amounts of the intermediate were processed to mature-size
LasA protease. The addition of TLCK, on the other hand, blocked the
extracellular processing of proLasA almost completely. Virtually no
increase in the amount of the intermediate beyond that seen at 60 min
was evident, and only a slight increase in the level of mature-size
LasA protease was seen, which most likely reflected the action of
alkaline proteinase. Furthermore, whereas the proLasA level decreased
gradually with time in the control samples, a slight accumulation of
proLasA was noted in the media from TLCK-treated cultures. TLCK seemed
to affect the secretion of proLasA. However, immunoblotting with
antibodies to the lysine-specific endopeptidase revealed that TLCK had
no effect on the secretion of this enzyme (data not shown). Activity
assays with the specific substrate, Tosyl-Gly-Pro-Lys-para nitroanilide
(5), indicated that TLCK blocked the activity of the secreted
lysine-specific endopeptidase by at least 95%. This inhibition most
probably accounted for the observed drop in the rate of processing of
secreted proLasA, supporting a role for the lysine-specific
endopeptidase in the physiological processing of proLasA. We conclude
that elastase and the lysine-specific protease are key players in the
extracellular processing of proLasA in P. aeruginosa. In
their absence, alkaline proteinase may substitute for their action,
albeit not as effectively and via a mechanism that does not involve the
formation of distinct intermediates.
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Fig. 4.
Extracellular processing of proLasA in
P. aeruginosa strain FRD740 is inhibited in the presence of
TLCK. Late-log cells of P. aeruginosa strain FRD740
were suspended in fresh medium and incubated for 60 min at 37 °C,
and then the cell suspension was split into two portions. One of these
was made 2 mM in TLCK, whereas the other was supplemented
with an equal volume of fresh medium (control); both were incubated for
an additional 90 min. Aliquots removed at 30-min intervals throughout
the experiment were concentrated with TCA and analyzed by SDS-PAGE and
immunoblotting with antibodies to LasA protease. Proteins in 10-fold
concentrated samples corresponding to 170 µl of the original cell
culture were applied on each lane. Left, molecular masses in
kDa. The faint band above that of the 28-kDa intermediate detected in
lanes 90 to 150 (min) of the control culture is
an artifact resulting from a slight movement of the blot against the
film during a very short (15 s) exposure.
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Fig. 5.
Secretion of the elastase propeptide.
Samples (25 µl each) of culture supernatants of P. aeruginosa FRD2 from the experiment presented in Fig. 1 were
analyzed (without prior TCA precipitation of the medium proteins) by
SDS-PAGE and immunoblotting with antibodies to elastase (top
panel) or its propeptide (bottom panel). Ela
and Pro, migration positions of purified elastase and its
propeptide, respectively. Top, time (minutes after medium
replacement and reequilibration to 37 °C) at which each sample was
taken. Left, molecular masses in kDa.
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Fig. 6.
Elastase is secreted as a noncovalent complex
with its propeptide. P. aeruginosa strain FRD2 was grown as
described in the legend to Fig. 1. Protease inhibitors were added to
the supernatants, which were then subjected to PAGE and immunoblotting
under nondenaturing conditions. Shown are blots with antibodies to
elastase (left panel) and its propeptide (right
panel). Top, time (minutes after medium replacement and
reequilibration to 37 °C) at which each sample was taken.
Ela, elastase. Lanes Ela and E/P were
loaded with 10 and 15 ng of purified elastase and purified periplasmic
elastase-propeptide complex (14), respectively. The differences in the
pattern of the E/P band as a function of time when probed with elastase
(left panel) and propeptide (right panel)
antibodies reflect differences in the strength of these antibodies.
Visualization of E/P with the propeptide antibody required a high
antibody concentration and prolonged exposure of the film beyond the
linear range of antigen detection.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-lytic protease from
Lysobacter enzymogenes, another protease that depends on its
propeptide for folding and secretion, has been demonstrated in the
medium of E. coli cells expressing the -lytic protease gene (28). However, a limitation of this study was the use of a
heterologous organism, E. coli, as a host. Because the genes for the E. coli type II export machinery (gsp)
are barely expressed (29), the appearance of extracellular -lytic
protease and its propeptide in the medium of these cells could result
from the perturbation of membrane physiology caused by overexpression
of the -lytic protease. No evidence for the secretion of
amino-terminal propeptides of other proteases has yet been obtained in
the natural bacterial host. In the case of P. aeruginosa,
standard culture supernatants contain high levels of proteolytic
activity, precluding the survival of labile protein species such as
free propeptides and zymogens. To prevent the proteolytic degradation
of such short-lived proteins, protein secretion was analyzed in the
present study after replacing the culture broth with fresh medium to
examine the onset of protease production. In addition, proteolysis was controlled by adding either TCA or protease inhibitors to the samples.
Our results clearly indicate that elastase is secreted as a noncovalent
complex with its propeptide, and the propeptide is degraded
extracellularly, most likely by elastase itself. This is suggested by
the inverse relationship between the levels of elastase and its
propeptide in medium samples removed from P. aeruginosa
cultures at times later than 40 min (Fig. 5). The apparent stability of
the propeptide seen at the earlier time points is misleading. In fact,
it probably represents a steady-state situation in which the rate of
secretion and that of propeptide degradation are still balanced. The
complete disappearance of the propeptide observed once high amounts of
elastase accumulate in the medium (Fig. 5) strongly supports the
involvement of elastase in the degradation of its own propeptide.
Furthermore, in a previous study (15), we noted that after its
isolation from the periplasm, the elastase-propeptide complex becomes
extremely unstable, undergoing spontaneous activation due to the loss
of the propeptide, even when stored at low temperatures. Stabilization
of the purified inactive complex was achieved by adding a detergent,
Brij-35. The detergent probably substituted for the stabilizing
interactions of the elastase-propeptide complex with envelope
components, interactions that are lost upon purification as well as
after secretion. Extracellular degradation of the propeptide apparently
does not require prior dissociation of the complex, because native
intermediates recognized by antibodies to both elastase and its
propeptide were demonstrated (Fig. 6).
-lytic protease (31).
-lytic
protease from L. enzymogenes (28). It has been suggested that only properly folded and processed forms of such proteases are
exported into the extracellular environment (29). Our demonstration that elastase and LasA protease are both secreted with their
propeptides, the first experimental evidence for such a mechanism,
suggests that secretion of the propeptide may be a property shared by
other extracellular bacterial proteases. This also favors a role for the propeptides in targeting and recognition by the export machinery of
their cognate enzymes. However, autoprocessing of the propeptide may
not be a universal mechanism. This mechanism may be utilized by
endopeptidases with broad specificity, whereas proenzymes of endopeptidases with restricted specificity or those of exopeptidases may depend on heterologous endopeptidases for processing as well as the
ultimate degradation of their propeptides. Proenzymes of many
endopeptidases with general proteolytic activity, including subtilisin
E (32), -lytic protease (28), elastase (12, 13), thermolysin, and
other thermolysin-related neutral proteases (33, 34), have indeed been
shown to be processed autocatalytically, whereas procarboxypeptidase Y
depends on yeast vacuolar endopeptidases for processing (35).
Note Addded in Proof
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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