(Received for publication, December 2, 1996, and in revised form, January 13, 1997)
From the Maurice and Gabriela Goldschleger Eye
Research Institute, Tel-Aviv University Sackler Faculty of Medicine,
Sheba Medical Center, Tel-Hashomer 52621, Israel, the ¶ University
of Pennsylvania School of Dental Medicine, Biopolymer Analysis
Laboratory, Philadelphia, Pennsylvania 19104, and the
Department
of Microbiology and Immunology, University of Tennessee and Veterans
Administration Medical Center, Memphis, Tennessee 38163
LasA is an extracellular protease of
Pseudomonas aeruginosa that enhances the elastolytic
activity of Pseudomonas elastase and other proteases by
cleaving elastin at unknown sites. LasA is also a staphylolytic
protease, an enzyme that lyses Staphylococcus aureus cells
by cleaving the peptidoglycan pentaglycine interpeptides. Here we
showed that the staphylolytic activity of LasA is inhibited by
tetraethylenepentamine and 1,10-phenanthroline (zinc chelators) as well
as excess Zn2+ and dithiothreitol. However, LasA was not
inhibited by several serine or cysteine proteinase inhibitors including
diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, leupeptin,
and N-ethylmaleimide. LasA staphylolytic activity was also
insensitive to
N-p-tosyl-L-lysine
chloromethyl ketone or phosphoramidon. EDTA and EGTA were inhibitory
only at concentrations greater than 20 mM. Without added
inhibitors, LasA obtained by DEAE-cellulose fractionation was active
toward
-casein, but the same cleavage patterns were observed with
column fractions containing little or no LasA. The
-casein cleaving
activity was fully blocked in the presence of inhibitors that did not
affect staphylolytic activity. In the presence of such inhibitors,
purified LasA was inactive toward acetyl-Ala4 and
benzyloxycarbonyl-Gly-Pro-Gly-Gly-Pro-Ala, but it degraded soluble
recombinant human elastin as well as insoluble elastin. N-terminal
amino acid sequencing of two fragments derived from soluble elastin
indicated that both resulted from cleavages of Gly-Ala peptide bonds
located within similar sequences, Pro-Gly-Val-Gly-Gly-Ala-Xaa (where
Xaa is Phe or Gly). In addition, Ala was identified as the predominant
N-terminal residue in fragments released by LasA from insoluble
elastin. A dose-dependence study of elastase stimulation by LasA
indicated that a high molar ratio of LasA to elastase was required for
significant enhancement of elastolysis. The present results suggest
that LasA is a zinc metalloendopeptidase selective for Gly-Ala peptide
bonds within Gly-Gly-Ala sequences in elastin. Substrates that contain
no Gly-Gly peptide bonds such as
-casein appear to be resistant to
LasA.
Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, produces several extracellular proteolytic enzymes that are thought to play a role in the pathogenesis of this organism. Most studies of P. aeruginosa proteinases (for reviews, see Refs. 1-4) have emphasized three extracellular endopeptidases, elastase (also termed pseudolysin), alkaline proteinase, and LasA. A lysine-specific endopeptidase (5) and an endopeptidase that lyses Staphylococcus aureus cells by cleaving the pentaglycine bridges within their cell wall peptidoglycan network have also been described (6, 7). Of all these endopeptidases, elastase is generally the most abundant. Furthermore, it displays a remarkably broad substrate specificity and is capable of degrading many host proteins. This includes elastin, a major component of connective tissues that is resistant to hydrolysis by most proteases (1, 2). Elastase is therefore regarded as one of the major virulence factors of P. aeruginosa. Alkaline proteinase exhibits a broad cleavage specificity but is not as potent as elastase and has no elastolytic activity (1).
LasA was first identified by a mutation (lasA1) in strain
PAO1 that resulted in a marked reduction in the ability of the bacteria to degrade elastin (8). Subsequent characterization of the lasA gene indicated that it encodes a 40-kDa protein
homologous to the -lytic endopeptidase of Lysobacter
enzymogenes (9). Culture supernatants of defined mutants
inactivated in lasA were deficient in elastolytic activity,
but this activity was restored by the addition of LasA produced in
Escherichia coli (9, 10). Peters and Galloway (11) isolated
a 22-kDa LasA fragment from culture filtrates of P. aeruginosa that enhanced elastolytic activity of
Pseudomonas elastase as well as that of other proteases.
Subsequently, elastase-negative mutants of P. aeruginosa
were found to retain some elastolytic activity (12, 13), and the
purified LasA fragment was shown to possess basal elastolytic activity
(14). The 40-kDa product of lasA was identified as a
precursor of the extracellular LasA protein (15), and because of its
action on elastin, the secreted 22-kDa form of LasA has been referred
to as a second elastase (12-14).
We found that LasA has staphylolytic activity and demonstrated LasA
identity with the long recognized (6, 7) staphylolytic endopeptidase of
P. aeruginosa (16). This, along with the homology to
L. enzymogenes and Achromobacter lyticus
-lytic endopeptidases (4, 16), placed LasA in the
-lytic
endopeptidase family of proteases and raised the possibility that LasA
may enhance elastolysis by cleaving abundant Gly-Gly peptide bonds in
elastin.
LasA has been reported to cleave -casein at the
Lys29-Ile30 peptide bond (14). This cleavage
seemed inconsistent with the apparent affinity of LasA for Gly-Gly
peptide bonds. In addition, the inhibition pattern reported for this
-casein cleavage was incompatible with that found for LasA
staphylolytic activity. Inhibitors of the
-casein-cleaving activity
(DFP,1 PMSF, and TLCK) had no effect on the
staphylolytic activity of LasA, and 1,10-phenanthroline, an inhibitor
of LasA staphylolytic activity, did not block
-casein degradation
(17). Hence, LasA was proposed to be a "modified" serine protease
(14, 17).
Here we present evidence that LasA is a zinc metalloendopeptidase that
preferentially cleaves peptide bonds subsequent to Gly-Gly pairs in
proteins and peptides. We identify Gly-Gly-Ala sequences as the
preferred sites of LasA cleavage in elastin and show that the ability
of LasA to enhance elastin digestion by elastase largely depends on the
amount of elastase and may require excess amounts of LasA. Finally, we
provide evidence suggesting that -casein digestion by LasA is only
due to a contaminating protease.
LasA and elastase were purified from P. aeruginosa strains FRD2 and Habs serotype 1, respectively. Both strains were grown at 37 °C for 24 h in tryptic soy broth without dextrose (Difco), and the enzymes were purified from the respective cell-free conditioned media by ammonium sulfate precipitation and DEAE-cellulose chromatography as described (16, 18). LasA eluted first from the column (peak I) and was followed by three additional protein peaks designated II, III, and IV. The major constituents in peaks II and III migrated as proteins of about 23 and 28 kDa, respectively. Elastase was recovered in peak IV (16).
Assay of Staphylolytic ActivityStaphylolytic activity was determined spectrophotometrically by monitoring the decrease in absorbance at 595 nm of a heat-killed S. aureus cell suspension (0.3 mg/ml; 0.02 M Tris-HCl, pH 8.5 (buffer A)) (16). The reaction volume was 1 ml, and the amount of purified LasA added was 0.4-0.7 µg (0.05-0.09 units). One unit of activity was defined as the amount of enzyme that causes an A595 decrease of 1 absorbance unit/min. For inhibition studies with this assay, LasA was preincubated (30 min, room temperature) with the specified inhibitor, and then the assay reaction was performed in the presence of the inhibitor.
Assay of Elastolytic ActivityElastolytic activity was determined with insoluble elastin-Congo red (Sigma) as the substrate. Reaction suspensions (1.1 ml in 50 mM Tris-HCl, 0.5 mM CaCl2, 0.2 mg/ml bovine serum albumin, pH 7.5) containing 10 mg of elastin-Congo red and 0.5 µg of elastase were incubated at 37 °C for 2 h with or without varying amounts of LasA that was first treated with PMSF (0.4 mM) and TLCK (5 mM). Reactions were stopped by adding 0.1 ml of 120 mM EDTA followed by immediate cooling and centrifugation (Beckman Microfuge, 4 min). The degree of elastin solubilization was determined by measuring the absorbance at 495 nm of the clear supernatant.
LasA Digestion of ElastinElastin digests with LasA were
performed in 0.02 or 0.05 M Tris-HCl, pH 8.5, containing 5 mM TLCK, 0.4 mM PMSF, and 1 mM
phosphoramidon (TI buffers). LasA was incubated (30 min, room
temperature) with the inhibitors before mixing with the elastin
substrate. Soluble recombinant human elastin (tropoelastin, rTE26a,
20 µg in 25 µl of 0.02 M TI buffer) (19) was mixed with
LasA (56 ng in 20 µl of 0.02 M TI buffer) and incubated
for 2 h at 37 °C. The reaction was terminated by heating in SDS
sample buffer (20), and the products were analyzed by SDS-PAGE in a
12% polyacrylamide gel. For LasA digests of insoluble elastin, 5 µg
of LasA were preincubated (30 min, room temperature) in 250 µl of
0.05 M TI buffer. 2.5 mg of solid insoluble elastin were
then added, and the resulting suspensions were incubated at 37 °C
for 8 h. The reactions were stopped by centrifugation (as above)
to remove remaining insoluble elastin, and the supernatants were heated
in SDS sample buffer. Reaction products were analyzed by SDS-PAGE in a
composite gel in which the lower 3 cm of the separating gel contained
12% acrylamide and the upper 6 cm of the gel contained 6%
acrylamide.
LasA (50 ng in 25 µl of
buffer A) was mixed with -casein (Sigma, 7 µg in
5 µl of buffer A), and the solutions were incubated for 3 h at
37 °C. The reaction was terminated by heating (100 °C, 2 min) in
SDS sample buffer, and the products were identified by SDS-PAGE in 14%
polyacrylamide gels (20). Protein bands were visualized by Coomassie
Blue staining. For comparison,
-casein was similarly digested with 5 ng (or as otherwise specified) of protein from fraction II (peak II,
eluted from the DEAE-cellulose column following the LasA peak I) (16).
For inhibition studies, each enzyme sample was preincubated (1 h, room
temperature) with the specified inhibitor(s), and the reaction was
initiated by adding
-casein substrate solution containing the
respective inhibitor(s).
Protein fragments for N-terminal sequence analysis were separated by SDS-PAGE and then electroblotted to Immobilon-P (Millipore Corp.) membranes (21). Proteins were identified by rapid staining with Coomassie Blue (22) and subjected to automated Edman degradation with an Applied Biosystems 477A protein sequencer.
The finding that LasA possesses high staphylolytic activity (16) provided a simple and specific assay method with which to determine LasA enzymatic activity. We took advantage of this to study the inhibition properties of LasA. Table I shows that strong zinc chelators such as tetraethylenepentamine and 1,10-phenanthroline effectively inhibited the staphylolytic activity of LasA. Non-chelating analogs of 1,10-phenanthroline, 4,7- and 1,7-phenanthroline, had no effect on the staphylolytic activity. ZnCl2, at concentrations higher than 10 µM, was inhibitory to the enzyme. Relatively high concentrations (25 mM) of EDTA and EGTA were required for LasA inhibition, suggesting that LasA may not require calcium ions for either activity or stability. Dithiothreitol inhibited LasA, most likely due to reduction of critical disulfide bonds. Staphylolytic activity of LasA was insensitive to the serine protease inhibitors DFP and PMSF, and it was not blocked by TLCK, N-ethylmaleimide, leupeptin, soybean trypsin inhibitor, or phosphoramidon. Together, these results strongly suggest that LasA is a zinc-dependent metalloendopeptidase.
|
To define the cleavage
sites of LasA in elastin, recombinant tropoelastin lacking exon 26 (rTE26a) (19) was incubated with LasA under conditions yielding a
limited number of elastin fragments. To prevent potential nonspecific
cleavages, LasA was pretreated with a mixture of protease inhibitors
(TLCK, PMSF, and phosphoramidon) that do not affect its staphylolytic
activity, and the digest was performed in the presence of the same
inhibitors. The tropoelastin substrate (Fig.
1A, lane 1, molecular mass ~65
kDa) was degraded into four distinct fragments, I to IV, with apparent
molecular masses of 61, 49, 40, and 25 kDa, respectively (Fig.
1A, lane 2). The amino acid sequences of the
first 8 and 7 residues of fragments II and III were determined by
automatic Edman degradation and identified as AFAGIPGV and AGVPGVP,
respectively. These apolar sequences correspond to positions 184-191
(fragment II) and 279-285 (fragment III) in rTE
26a, and in both
instances they follow an identical sequence, PGVGG. Thus, both
fragments resulted from the cleavage of a Gly-Ala bond in a Gly-Gly-Ala
sequence located within a stretch of nonpolar amino acid residues. No
sequences were obtained for fragments I and IV.
Insoluble elastin was also digested with LasA, and SDS-PAGE analysis of the solubilized material revealed three main heterogeneous populations of molecules with approximate sizes in the ranges >200 kDa, 70-110 kDa, and 20-70 kDa (Fig. 1B, lane 1). Results with elastin-Congo red as the substrate were similar although the solubilized products in this instance showed greater variability in size (Fig. 1B, lane 2). The N-terminal residues of elastin fragments in the three size groups derived from unmodified elastin were determined by automatic Edman degradation. In all three instances, Ala was found as the predominant N-terminal amino acid residue although small amounts of Gly and Phe were also detectable. To exclude the possibility that the high levels of alanine found in the first cycle of the Edman degradation resulted from cleavages of peptide bonds within the relatively long stretches of alanine residues found in elastin, we tested the effect of LasA on acetyl-Ala4, a synthetic substrate that is readily degraded by P. aeruginosa elastase. To prevent potential cleavages by traces of elastase, the digest was performed in the presence of phosphoramidon. Thin layer chromatography analysis revealed that LasA has no effect on Ac-Ala4 (data not shown). The numerous Gly-Gly-Pro sequences in elastin are apparently not cleaved by LasA, a conclusion supported by our observation that the synthetic collagenase substrate, benzyloxycarbonyl-GPGGPA, was resistant to LasA (data not shown). The low levels of Gly and Phe detected in the first Edman degradation cycle may reflect cleavages of some Gly-Gly and Gly-Phe peptide bonds.
LasA Enhancement of Elastase ActivityLasA has been shown to
enhance the elastolytic activity of Pseudomonas elastase
in a dose-dependent manner (11). However, the effect has
only been shown with high elastase input (10 µg) and a relatively
narrow range of LasA/elastase molar ratios (0.1 to 1.7, 0.5-10 µg of
LasA). Here we extended these studies using a low elastase input (0.5 µg) and a broad range of LasA/elastase molar ratios (0.1 to 80, 0.03-24 µg of LasA). To stabilize the enzymes, all incubations were
performed in the presence of 0.2 mg/ml bovine serum albumin. Controls
to ascertain the stability of elastase and LasA in the presence of each
other included mixtures of LasA and elastase (0.5 µg) at
representative LasA/elastase molar ratios of 1:1 and 10:1. These
mixtures were incubated without elastin. In addition, each enzyme was
incubated alone. At the end of the incubation, proteolytic activity of
elastase (versus azocasein (16, 18)) and staphylolytic
activity of LasA (measured in the presence of 0.1 mM
phosphoramidon to prevent interference by elastase) were determined,
and no loss in activity of either enzyme was noted whether incubated
alone or in combination with each other. Thus, degradation of one
enzyme by the other in the assays of combined elastolytic activity is
unlikely. In the assays of elastolytic activity, LasA alone was about
30-fold less effective than elastase in solubilizing the elastin-Congo
red substrate. When added to elastase (Fig. 2),
essentially no increase in elastolytic activity was observed at
LasA/elastase ratios lower than 1 (LasA input of 0.03-0.3 µg). A
2-fold enhancement was obtained at a LasA/elastase molar ratio of 5 (1.5 µg of LasA), whereas 30- and 80-fold excesses of LasA (9 and 24 µg of LasA) were required to obtain 4- and 12-fold enhancement of
elastolysis, respectively. These results confirm the limited
elastolytic power of LasA alone and show that, at a low elastase
concentration, excessive amounts of LasA are required to significantly
increase the rate of solubilization of insoluble amorphous elastin.
LasA has been reported to
cleave -casein into two distinct fragments by cleavage at the
Lys29-Ile30 peptide bond (14, 17). Our LasA
preparation, which eluted as fraction I on a DEAE-cellulose column
(16), initially exhibited a similar activity toward
-casein (Fig.
3A, lane 2). However, the same
degradation pattern was observed with DEAE-cellulose fractions II and
III, which eluted from the DEAE-cellulose column after LasA and showed
very little or no staphylolytic activity (16). Moreover, fractions II
and III were more effective in degrading
-casein than was the LasA
fraction (compare lanes 10 and 14 containing 5 and 2.5 ng of fraction II protein, respectively, with lane 2 containing 50 ng of the LasA fraction; similar data, obtained with 1 ng
of fraction III, are not shown). This raised the possibility that the
-casein degradation associated with this LasA preparation could
reflect the activity of a contaminating protease(s) rather than LasA
itself. To explore this possibility, we compared the LasA fraction with
fraction II in the hydrolysis of
-casein and its inhibition. To
achieve approximately the same level of activity toward
-casein with
both enzyme samples, the input of protein was 10-fold higher with the
LasA fraction than with fraction II (50 and 5 ng, respectively). As
shown in Fig. 3A (lanes 3 and 11),
-casein cleavage by both proteinase fractions was totally abolished
in the presence of a mixture of inhibitors containing phosphoramidon
(PHSN, a specific inhibitor of P. aeruginosa elastase), PMSF (a general serine protease inhibitor), TLCK (an inhibitor of lysine-specific serine proteases), and 1,10-phenanthroline (PHEN, an inhibitor of LasA as well as elastase). Since the
observed cleavage of
-casein could potentially represent the
combined effect of a variety of proteases, we compared subsets of this inhibitor mixture for the ability to block degradation. The inhibition profiles obtained with both the LasA fraction and fraction II under all
conditions were the same (Fig. 3A). Inhibition of both was
clearly dependent on TLCK, and omitting any of the other three inhibitors did not appear to affect the ability of TLCK to inhibit
-casein hydrolysis (lanes 4, 6, and
7 for LasA fraction I and lanes 12,
15, and 16 for fraction II). In the absence of
TLCK, the cleavage was essentially the same as that seen in the
controls (compare lane 2 with lanes 5 and
8; compare lane 10 with lanes 13 and
17). PMSF at 0.4 mM was not inhibitory and could
not substitute for TLCK (lanes 5, 13, and
14). However, in a subsequent experiment in which the effect
of each inhibitor on
-casein degradation by fraction II was tested
independently of the other inhibitors (Fig. 3B), PMSF at 2 mM not only successfully replaced TLCK as an inhibitor of
the reaction, but it even appeared to be more effective than TLCK in
blocking this activity (Fig. 3B, compare lanes 3 and 5). DFP (5 mM) was also highly inhibitory
(Fig. 3B, lane 4), and in agreement with the
results of Fig. 3A, neither 1,10-phenanthroline nor
phosphoramidon (Fig. 3B, lanes 6 and
8, respectively) alone inhibited fraction II action on
-casein. The same results were observed with LasA and fraction III
as the active fractions (data not shown). Together, these results
exclude
-casein as a LasA substrate and suggest that the observed
cleavages of
-casein arose from the action of a serine protease(s)
that was not fully resolved from LasA during purification on
DEAE-cellulose. To identify the peptide bonds cleaved by this putative
serine protease,
-casein fragments 1 and 2 (Fig. 3C),
which were generated by fraction II in the presence of
1,10-phenanthroline, phosphoramidon, and PMSF were subjected to
automatic N-terminal amino acid sequence analysis. The sequences of the
first 10 and 4 residues of fragments 1 and 2 were found to be
XELEELNVPG and IEXF, respectively (X, unidentified residue). These sequences correspond to residues 1-10 and
30-33 in
-casein (23). Thus, fragment 1 represents an N-terminal
-casein peptide that most likely resulted from cleavage(s) of a
peptide bond(s) within the C-terminal portion of the
-casein
molecule, whereas fragment II apparently resulted from cleavage of the
Lys29-Ile30 of
-casein, the same as that
previously reported for the LasA cleavage site (14, 17).
Previous conflicting results regarding the classification and
cleavage specificity of LasA (14, 17) led us to the present study of
these issues. To assign LasA into one of the four known classes of
proteases, serine, aspartic, thiol, or metalloendopeptidases (24), we
examined the inhibition properties of the enzyme (Table I). The assay
of staphylolytic activity that we used for this purpose is sensitive
and specific. Although the rate of staphylolysis by LasA may be
increased by up to 2.5-fold in the presence of P. aeruginosa
elastase or alkaline proteinase (observed by us (15) and others (17)),
neither elastase nor alkaline proteinase alone show staphylolytic
activity. In addition, for significant enhancement of LasA action on
S. aureus cells, relatively large amounts of elastase or
alkaline proteinase are required. As suggested from results with
-casein as the substrate, our purified LasA preparation apparently
contained traces of contaminating proteases. These, however, are below
the levels that may affect LasA action on the staphylococcal cells.
Thus, the assay of staphylolytic activity as used here is reliable.
Since LasA staphylolytic activity was inhibited by two zinc chelators
(1,10-phenanthroline and tetraethylenepentamine) but not by any of the
other class-specific inhibitors, we conclude that LasA is a
zinc-dependent metalloendopeptidase. This conclusion is
further supported by the inhibitory effect of excess zinc ions (Table
I), a property characteristic of other zinc-dependent peptidases (reviewed in Ref. 25).
The amino acid sequence of LasA shows approximately 40% identity with
those of L. enzymogenes and A. lyticus -lytic
endopeptidases (4, 16, 26, 27). In addition, the sequence of the first 40 amino acid residues of a recently described Aeromonas
hydrophila zinc-dependent endopeptidase (AhP) (28) is
46 and 69% identical with those of L. enzymogenes
-lytic
endopeptidase and LasA, respectively. Thus, LasA, AhP, and the
-lytic endopeptidases of L. enzymogenes and A. lyticus are closely related. The
-lytic endopeptidase of
L. enzymogenes (26) and AhP (28) were shown to contain 1 zinc atom/mol. This supports LasA as a zinc metalloendopeptidase and is
consistent with the recent demonstration that efficient production of
LasA by P. aeruginosa requires zinc ions (29). LasA as well
as the Lysobacter and Achromobacter
-lytic
endopeptidases (15, 26, 27) does not contain the HEXXH
zinc-binding motif typical of most zinc proteinases. An HXH
motif noted in A. lyticus
-lytic endopeptidase (27) and
shared with both the Lysobacter enzyme and LasA (positions
120-122) was proposed as a potential zinc ligand (27). Substitution in
LasA of His-120 blocks LasA activity (15), suggesting that His-120 may
be one of the zinc ligands in LasA.
As a zinc metalloendopeptidase, LasA action on substrates other than
S. aureus is expected to be blocked by zinc chelators but
not by inhibitors of the other classes of proteases. Our results with
soluble tropoelastin and insoluble elastin as substrates were fully
consistent in this regard. While LasA action on elastin was inhibited
by 1,10-phenanthroline (data not shown), it was not blocked by
phosphoramidon nor was it affected by DFP, PMSF, or TLCK. Our
identification of the preferred LasA cleavage sites in elastin as the
Gly-Ala peptide bonds within the Gly-Gly-Ala sequences surrounded by
apolar sequences is in accordance with previous reports of
LasA/staphylolytic protease specificity of cleavage, i.e.
hydrolysis of pentaglycine, hexaglycine, or oligopeptides containing at
least 2 glycine residues in a row (6, 7, 16, 17). That Gly-Gly-Ala
sequences are authentic LasA cleavage sites in elastin is further
supported by analogy to AhP substrate specificity (28). AhP is
selective for substrates with (internal) Gly-Gly or acetyl-Gly in the
P2 and P1 (30) positions, respectively, with
nonpolar amino acid residues in the P1 and P2
positions favored. Synthetic peptide derivatives with Gly-Gly pairs
followed by Phe or Ala residues at the P1
position are
among the best synthetic substrates for AhP, and this endopeptidase
also readily hydrolyzes the Gly-Ala bond within a Gly-Gly-Ala sequence
in the
-chain dimer of fibrin (28). Although at a slower rate, AhP also cleaves the Gly-Gly bond in acetyl-Gly-Gly-Phe-amide and the
respective Ala-amide derivative (28). Thus, our demonstration of Ala as
the prevalent N-terminal residue in mixtures of fragments released by
LasA from insoluble elastin and the finding of low levels of glycine
and phenylalanine in the first position of such fragments are also
consistent with AhP specificity of cleavage. This suggests Gly-Phe and
Gly-Gly as additional sites of LasA cleavage in elastin. Such cleavages
are further supported by early reports demonstrating cleavages by
L. enzymogenes
-lytic endopeptidase of Gly-Phe peptide
bonds in the insulin B chain and in the synthetic substrate
benzyloxycarbonyl-Gly-Phe-amide (31, 32). The number of Gly-Gly pairs
followed by Ala, Gly, or Phe in elastin is relatively small. This may
explain the limited power of LasA as an elastase.
Our results on LasA inhibition properties and cleavage specificity with
elastin as the substrate are in agreement with the current body of
evidence for the LasA-related -lytic proteases (4). The recent
reports showing
-casein as a substrate for LasA, with a Lys-Ile
peptide bond as a major site of cleavage and inhibition of this
cleavage by TLCK, PMSF, and DFP (14, 17), caught our attention because
we would not have predicted this. As shown here, LasA preferentially
cleaves peptide bonds subsequent to Gly-Gly pairs, in particular those
followed by Ala and located within apolar environments. However, no
such sequences exist in
-casein. Furthermore, the only 4 Gly
residues in
-casein are followed by Pro, Glu, or Val (23), and these
sites are unlikely to be sensitive to LasA. In addition, the Lys-Ile
bond of
-casein is situated within a highly charged environment
containing a Glu, a phosphorylated Ser, and 2 Lys residues in close
vicinity (23). Charged residues should interfere with LasA action, as
was shown directly for AhP (28), which is a protease similar to LasA. Yet our initial studies did show that the LasA preparation could cleave
-casein with inhibition by TLCK, PMSF, and DFP. This would suggest
that the active entity is a lysine-specific serine proteinase rather
than a zinc metalloendopeptidase such as LasA. Further investigation
revealed that fractions II and III of the DEAE-cellulose fractionation
of P. aeruginosa extracellular proteins, which contained only traces of LasA, were even more effective than the LasA fraction in
eliciting this cleavage. A 30-kDa lysine-specific endopeptidase from
P. aeruginosa has been described that cleaves peptide bonds involving the carboxyl side of internal lysine residues and is inhibited by TLCK (5). Our fraction III contained a protein of about 28 kDa molecular mass as a major component (16), and this may be the
lysyl-specific protease that was responsible for the observed
-casein cleaving activity in LasA preparations.
Several studies have demonstrated a synergism between P. aeruginosa elastase and LasA protease in the efficient degradation of elastin. Although purified LasA alone does not show high elastolytic activity, culture filtrates of lasA mutants (e.g. PAO-E64 and FRD2128) show much less elastolytic activity (30-90%) when compared with their respective wild type strains (8, 10, 11, 16). Peters and Galloway (11) examined this further with purified components and showed that the elastolytic activity of elastase (10 µg input) increased 25-fold in the presence of purified LasA (10 µg input), which is a LasA/elastase molar ratio of ~1.7. We also used purified enzymes to characterize the LasA-dependent enhancement of elastolytic activity but at a 20-fold lower elastase input as this amount is more in the range used in our standard assays of elastolytic activity. Under such conditions, LasA appears to be much less effective in potentiating elastase activity. Although the degree of enhancement of elastolysis was still dependent on LasA concentration, a LasA/elastase molar ratio of ~30 was required to increase elastolysis by 4-fold, and a molar ratio of ~80 increased the rate of elastolysis by 12-fold. This quantitative difference between situations with high and low elastase concentrations demonstrates that the synergistic effect of LasA and elastase in the solubilization of insoluble, amorphous elastin substrates is better pronounced in the presence of high elastase concentrations. This may be explained by the complementary specificities of LasA and elastase, the insoluble nature of the elastin substrate, and the fact that only soluble degradation products are apparent in the assay. The limited cleavages by LasA, either alone or in the presence of small amounts of elastase, may only yield a limited number of soluble elastin fragments. On the other hand, in the presence of excess elastase many insoluble elastin fragments may be produced in which LasA cleavage sites are likely more accessible to LasA than in the intact elastin substrate, and thus, even small amounts of LasA may largely increase the rate of elastin solubilization. Colonies of lasA mutants on elastin-containing agar plates are dramatically deficient in elastin clearing when compared with wild type strains (8, 10), which suggests that the relative concentration of elastase is high under these conditions. We conclude that elastase potentiates LasA hydrolysis of elastin and is critical for LasA enhancement of the elastolytic potential of P. aeruginosa.
The limited elastolytic power of LasA suggests that elastin may not be
the primary or only substrate for LasA action. Collagen, another
glycine-rich connective tissue protein that we examined as a potential
substrate for LasA, was also a poor substrate. Only a minor fraction of
the -chains was partially cleaved and no more than three smaller
fragments were generated even after prolonged incubations (data not
shown). These limited cleavages could occur at sequences such as
Gly-Ala-Ala-Gly found at several sites within the
1(I) and
2(I)
collagen chains (33). LasA was reported to effectively hydrolyze two
biologically active peptides containing internal Gly-Gly or Gly-Gly-Gly
sequences (17). Although consistent with LasA specificity, cleavage of these peptides (speract, a stimulator of sea urchin spermatozoa, and a
sleep-inducing peptide, respectively) has no immediate relevance to
P. aeruginosa infections. LasA, however, is a potent
staphylolytic protease. By virtue of its action on the cell wall
peptidoglycan of S. aureus cells, LasA may provide P. aeruginosa with a selective advantage against S. aureus
cells during colonization at the infection site.
We gratefully acknowledge Jerry Seyer at the Veterans Administration Research Service, Memphis for the N-terminal sequence analyses.