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J. Biol. Chem., Vol. 277, Issue 13, 11042-11049, March 29, 2002
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From the Universitätsklinikum Hamburg-Eppendorf, D-20246
Hamburg, Germany,
Received for publication, October 2, 2001, and in revised form, December 26, 2001
Stenotrophomonas maltophilia is
increasingly emerging as a multiresistant pathogen in the hospital
environment. In immunosuppressed patients, these bacteria may cause
severe infections associated with tissue lesions such as pulmonary
hemorrhage. This suggests proteolysis as a possible pathogenic
mechanism in these infections. This study describes a protease with
broad specificity secreted by S. maltophilia. The gene,
termed StmPr1, codes for a 63-kDa precursor that is
processed to the mature protein of 47 kDa. The enzyme is an alkaline
serine protease that, by sequence homology and enzymic properties, can
be further classified as a new member of the family of subtilases. It
differs from the classic subtilisins in molecular size, in substrate
specificity, and probably in the architecture of the active site. The
StmPr1 protease is able to degrade several human proteins
from serum and connective tissue. Furthermore, pan-protease inhibitors
such as Stenotrophomonas maltophilia, formerly referred to as
Xanthomonas maltophilia or Pseudomonas
maltophilia (1, 2), is an aerobic nonfermentative Gram-negative
bacterium of widespread occurrence. For healthy humans, it is regarded
as an opportunistic germ; it has been implicated in a variety of
infections without distinctive clinical features (for a review, see
Ref. 3). However, in immune-compromised patients, particularly those
with bone marrow aplasia or receiving intensive chemotherapy, cases of
fulminant hemorrhagic pneumonia have been reported, even with fatal
outcome (4-6). In patients not surviving infections with S. maltophilia, histological inspection of the lung tissue
revealed massive bleeding caused by damage to the lung epithelium (4).
There are further reports demonstrating involvement of this bacterium
in massive hemorrhagic processes of the small intestine and the
subclavian artery accompanied by severe lesions of the tissue (5, 6). These observations strongly suggest the participation of proteolytic activity, produced by the bacteria, which may damage the infected tissue. Indeed, it is known that members of the
Pseudomonaceae express and secrete a variety of proteases
(cf. Ref. 7). Whereas the primary function of these enzymes
is to provide a source of free amino acids for bacterial survival and
growth, there is accumulating evidence that bacterial proteases may
play a pathogenic role in the infected host by involvement in tissue
invasion and destruction, evasion of host defenses, and modulation of
the host immune system (8).
The broad administration of antibiotics currently applied in cases of
intensive care patients leads to selection of multiresistant S. maltophilia strains. Consequently, these bacteria are found with
increasing frequency in the hospital environment. Because of the known
multiresistance of this germ toward conventional antibiotics (for a
review, see Ref. 9), bacterial proteases involved in the pathogenesis
of human diseases are potential targets for specific drug development.
This prompted us to test cultures of S. maltophilia obtained
from patient material for the presence of proteolytic activity. Indeed,
a highly active protease was detected as a major secretion product of
the isolated bacteria.
This study describes the purification, cloning, and characterization of
the S. maltophilia extracellular protease.
Source and Cultivation of Bacteria--
S.
maltophilia was isolated from a bronchoalveolar lavage performed
on a patient at the Hamburg university clinic. The identity of
the germ was established by standard bacteriological techniques (API 20 NE; BioMérieux, Marcy-L'Etoile, France). Bacteria were grown
aerobically at 29 °C in a broth containing 5 µM
MnSO4, 0.36 mM CaCl2, 0.5 mM L-methionine, 0.8 mM
MgSO4, 2.2 mM K2HPO4,
3.7 mM KH2PO4, 6 mM
(NH4)2HPO4, 50 mM
disodium succinate, 2 g/liter yeast extract (ICN Biomedicals), 40 mg/liter gentamycin, 50 mg/liter cefotaxim, and 100 mg/liter ampicillin.
Purification of the Protease--
Cell-free supernatant (12.5 liters) was obtained from S. maltophilia cultures by
centrifugation at 4 °C and mixed with 80 ml of DE-52 (Whatman)
cellulose equilibrated with 10 mM Tris/HCl buffer, pH 7.4, and the mixture was stirred overnight at 4 °C. The matrix was then
collected by sedimentation, transferred into a column, and washed with
10 mM Tris/HCl buffer, pH 7.4. Protein fractions were
eluted by a linear gradient of 0-500 mM NaCl in the same
buffer at a flow rate of 2 ml/min. 30 fractions of 24 ml were collected
and assayed for proteolytic activity (see below). A single peak of
activity was detected; the respective fractions were pooled and
concentrated by ultrafiltration (Amicon YM 10 membrane) at 4 °C to a
final volume of 4 ml. This sample was divided into two aliquots, and
each was fractionated at a flow rate of 1 ml/min over a 310-ml column
of Fractogel EMD BioSec 650 (Merck) equilibrated with
phosphate-buffered saline. Fractions of 6 ml were collected, and the
two fractions containing most of the proteolytic activity were pooled
and served to characterize the protease. When this purified preparation
was compared with the crude bacterial supernatant, similar results were
obtained for enzyme action (kinetic parameters, stability, inhibitor
pattern, and salt dependence), indicating that the isolated protease is
intact and represents the major, if not only, protease produced by the bacteria.
Determination of StmPr1 Protein--
The protein concentration
in preparations of the native StmPr1 protease was determined
on SDS-polyacrylamide gels as described previously (40). Purified
recombinant StmPr1 protein, calibrated by the Biuret method
(11), served as a standard. The results were comparable with values
calculated from the optical density at 280 nm (mg/ml StmPr1
protease = 1.30 × A280).
Electrophoresis of Proteins--
SDS-PAGE was performed as
described in Ref. 12. Protein-containing samples were denatured with
10% trichloric acid before electrophoresis. Without this pretreatment,
additional bands of lower molecular mass appeared, obviously due
to self-digestion of the protease. Protein precipitates were collected
by centrifugation and washed with methanol to remove residual
trichloric acid. For autofluorography with a covalent inhibitor
specific for serine proteases, samples were incubated with 5 µCi of
[1,3-3H]diisopropylfluoro-phosphate (PerkinElmer Life
Sciences; 8.4 Ci/mmol) for 2 h at 37 °C, precipitated with 10%
trichloric acid, and subjected to SDS-PAGE. Polyacrylamide gels were
fixed with 10% acetic acid/30% methanol, equilibrated first with
water and then with 1 M sodium salicylate, dried, and
exposed to Kodak X-Omat film for 90 h at Enzyme Assays--
For the initial detection of proteolytic
activity in bacterial supernatants, a microassay using the nonspecific
chromogenic substrate azoalbumin (Sigma) was performed as described in
Ref. 13. In all other cases, a substrate specific for serine proteases was used (0.5 mM Suc-Ala-Ala-Pro-Phe-pNA, unless otherwise
stated). Hydrolysis was allowed to occur in 200 µl of 20 mM sodium phosphate, pH 9.0, containing 400 mM
NaCl at 37 °C. The amount of released p-nitroaniline
within initial time intervals was measured at 405 nm
( Protein Sequencing--
After SDS-PAGE, the protein was blotted
onto polyvinylidene difluoride membranes (Immobilon P; Millipore) and
stained with Coomassie Brilliant Blue R-250. The excised band was
sequenced by standard Edman degradation on an automated sequencer
(Applied Biosystems 476A). To obtain internal sequence information, the Coomassie Brilliant Blue R-250-stained protein band was cut out of the
SDS gel and in-gel digested with the endoproteases Lys-C or Asp-N
(Roche Molecular Biochemicals) in 50 mM Tris/HCl, pH 8.5, containing 1 mM EDTA (for digestion with Lys-C) or 50 mM Tris/HCl, pH 8.0 (for digestion with Asp-N), at 37 °C
overnight. The peptides obtained were separated by reverse phase
HPLC1 on a Vydac C4 column
(250 × 2.1 mm) at a flow rate of 200 µl/min. The following
gradient was applied: 2-80% B over a 50-min period (solvent A,
0.1% trifluoroacetic acid in water; solvent B, 0.085% trifluoroacetic
acid in 70% acetonitrile). The isolated peptides were sequenced by
Edman degradation following standard procedures.
Cloning of the StmPr1 Gene--
DNA oligomers were synthesized
complementary to the amino-terminal sequence PYYQQYQ and to the reverse
complement of the sequence APAAMRT obtained by digestion of the
purified protease with the endoprotease Lys-C (see above). Using these
primers (40 pmol each), amplification of chromosomal DNA (200 ng) from
S. maltophilia with Taq polymerase (Qiagen)
yielded a DNA fragment of 930 bp, which was sequenced (Applied
Biosystems 377). The sequence showed homology to known protease
sequences and served to design gene-specific primers. The rest of the
upstream and downstream portions of the gene were cloned by alternate
application of inverse PCR (14) using the EcoRII and
HinfI restriction sites and PCR using as template dA-tailed
fragments of genomic DNA generated by AatII, PstI, or SphI digestion, and one gene-specific
oligonucleotide plus poly-dT as primers. A final PCR product was
obtained using the Expand Long Template PCR System (Roche Molecular
Biochemicals), and primers comprising the identified start codon and
stop codon, respectively, were sequenced, cloned into the pGem-T Easy
vector (Promega), and resequenced for verification. The sequence of the StmPr1 gene has been deposited in the European Molecular
Biology Laboratory nucleotide sequence data base; the accession number is AJ291488.
Enzyme Hydrolysis of the Oxidized Insulin B
Chain--
Hydrolysis of the oxidized insulin B chain (Sigma) was
performed in 50 mM Tris/HCl buffer, pH 8.0, at room
temperature for 10 min and for 4 h. The reaction mixture (2 ml)
contained the enzyme and the substrate in a ratio of 1:200. The
peptides obtained after the enzymatic hydrolysis were separated by
reverse phase HPLC on a Vydac C18 column (125 × 2.1 mm) at a flow
rate of 200 µl/min, and the following gradient was applied: 2-65% B
over a 50-min period (solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.085% trifluoroacetic acid in 70% acetonitrile). The obtained peptides were identified by mass spectrometry on a hybrid tandem mass spectrometer (Qtof II; Micromass) equipped with a nanoelectrospray ion source. 10 µl of the collected fractions were
vacuum dried and redissolved in 5 µl of 60% methanol/5%
formic acid. 1 µl of this solution was transferred into a gold-coated nanoelectrospray needle (Micromass). From the masses of the peptides determined, a tentative assignment to fragments of the insulin B chain
was derived. The assignment was confirmed by subsequent tandem mass
spectrometric measurements of the peptide fragmentation pattern or, in
some cases, by Edman degradation.
Detection of Proteolytic Activity in Bacterial Cultures
A culture of S. maltophilia was grown from a specimen
of an immunocompromised patient. Proteolytic activity was detected in the cell-free growth medium of the bacterial culture using azo-albumin as an unspecific chromogenic substrate (data not shown).
To optimize bacterial cultures as a source for purification of the
putative protease, the production of the enzyme during culture growth
was measured. As shown in Fig. 1, the
proteolytic activity is hardly detectable in the early stages of the
culture; rather, protease production is up-regulated only toward the
end of the exponential phase of the growth curve. Proteolytic activity reached a maximum after about 22 h and remained unchanged for at
least 3 days.
Protease Purification
A culture supernatant of S. maltophilia served as a
source for the protease isolation. Adsorption on an anion exchange
resin was applied to concentrate and separate proteins from the
bacterial broth; elution by a salt gradient yielded a single peak of
proteolytic activity, which was then further fractionated by gel
filtration. SDS gel electrophoresis of the protease-containing fraction
revealed one major band of 47-kDa apparent molecular mass (Fig.
2A). Comparison with the
electrophoretic pattern of the crude bacterial supernatant indicated
that the 47-kDa protein represents the major secretory product of
S. maltophilia. Amino-terminal sequencing of the 47-kDa band
yielded the sequence LAPNDPYYQQ, which turned out to be absent from
protein sequence data bases. The sequence, however, showed homology
with the amino termini of several known bacterial proteases, the
closest of which is a serine protease from Dichelobacter
nodosus (15), a member of the family of subtilisin-like proteases
(cf. Ref. 7).
The Major Extracellular Protease of the Nosocomial Pathogen
Stenotrophomonas maltophilia
CHARACTERIZATION OF THE PROTEIN AND MOLECULAR CLONING OF THE
GENE*
,,,¶,
, and**
Institut für Medizinische
Biochemie und Molekularbiologie and § Institut für
Zellbiochemie und Klinische Neurobiologie
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-antitrypsin and 2-macroglobulin
were unable to abolish the activity of the bacterial protease. The
data support the interpretation that the extracellular protease
of S. maltophilia functions as a pathogenic factor and thus
could serve as a target for the development of therapeutic agents.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C.
405 = 9600 M
1
cm1). For determination of the IC50 of
protease inhibitors (antipain and chymostatin), assays contained 1.4 mM (Km) of the substrate
Suc-Ala-Ala-Pro-Phe-pNA and inhibitor over a wide range of
concentration. The IC50 is obtained as the constant
b in a nonlinear regression analysis of the function
(a/(b +10x)) when the reaction velocity
is plotted versus log10 of the inhibitor
concentration (x). Kinetic experiments with various
synthetic peptide p-nitroanilide substrates were carried out
in 100 mM Tris/HCl buffer, pH 8.2, at 25 °C and in the
presence of 5% dimethylformamide. The enzyme concentration was usually in the range of 1.95 × 108 to 9.12 × 109 M, and the concentration of the substrate
varied between 1.6 × 103 and 1.2 × 104 M. Kinetic parameters were calculated
from initial rate measurements of substrate hydrolysis using a
nonlinear regression analysis based on the function
(Vmax * x/(Km + x)), with x = the concentration of substrate.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (15K):
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Fig. 1.
Growth and production of extracellular
protease by S. maltophilia. 50 ml of culture
medium were inoculated with 0.1 ml of bacterial suspension (stationary
phase) and grown at 29 °C. Samples were taken at intervals and
tested for density ( 
) and, after centrifugation, enzyme activity
(
). Four separate experiments of this type were performed, yielding
similar results; data from one representative experiment are
presented.
View larger version (47K):
[in a new window]
Fig. 2.
SDS-PAGE of the StmPr1
protease. A, crude and purified fractions of
StmPr1 protease were analyzed on a 12% gel and stained with
Coomassie Brilliant Blue R-250. Lane 1, supernatant of
S. maltophilia (1.8 ml); lane 2, preparation of
purified StmPr1 protease (4 µg). B,
autofluorogram of [3H]diisopropyl fluorophosphate-labeled
proteins (see "Experimental Procedures"). Lane 1,
supernatant of S. maltophilia (0.2 ml); lane
2, preparation of purified StmPr1 protease (0.5 µg).
Covalent coupling with the radioactive inhibitor [3H]difluorophosphate confirmed that the 47-kDa protein of S. maltophilia is a serine protease (Fig. 2B). When the crude bacterial supernatant was allowed to react with the inhibitor, autoradiographs also showed mainly the 47-kDa band; the faint labeling of a lower molecular mass band may indicate a degradation product or the presence of another serine protease in trace amounts. Thus, the 47-kDa protein seems to be the enzyme mainly responsible for the extracellular proteolytic activity of S. maltophilia.
Sequence Determination by Molecular Cloning
To analyze the sequence of the protease and determine
its relationship to other proteases, the gene was cloned by PCR
techniques. Sequences from the amino terminus of the purified protein
and from an internal peptide (PLAPAAMRT) generated by Lys-C digestion served to design degenerate primers. Using genomic DNA of S. maltophilia as a template, a 930-bp amplified fragment was
obtained. Sequencing of the missing carboxyl-terminal portion of the
gene required additional steps of 3'-extension: PCR using
poly(A)-tailed fragments of genomic DNA as PCR template and inverse PCR
from highly diluted DNA fragments were applied until a stop codon was
found (Fig. 3).
|
Because it is common for most prokaryotic extracellular
proteases to be produced as larger precursor proteins (cf.
Ref. 7), 5'-extension of the DNA sequence was performed to obtain the
sequence of the entire gene. Applying the same techniques for extension as outlined above, a sequence was obtained that contained a stop codon
within the reading frame and only one ATG coding for a methionine in
position 132. However, two points argue against this ATG coding for
the translation initiation of the protease precursor: (i) it is not
preceded by a typical Shine-Dalgarno ribosome binding site, and (ii)
the sequence 3' to this ATG does not predict a signal peptide typical
for Gram-negative bacteria (16). Therefore, we assume that, following
an alternative bacterial codon usage, a GTG codes for translation
initiation, resulting in a methionine in position 150. In this case,
the synthax for both Shine-Dalgarno and signal sequences would be met
(Fig. 3). Further evidence for this GTG to function as a start codon
for the protease precursor came from the recombinant expression of the
gene. When the DNA starting with the GTG in question (and not with ATG
in position 132) was expressed in Escherichia coli, the
protein was correctly processed, resulting in the mature protease with
full enzymatic activity (data not shown). The DNA sequence of the gene
was further established, and the amino-terminal sequences of the
processed recombinant protein and of the native protease were found to
be identical. Moreover, antibodies generated against the native protein also recognized the recombinant gene product (data not shown).
Taken together, the open reading frame encodes a protein with a deduced
molecular mass of 63.0 kDa, corresponding to 618 amino acids in length
(Fig. 3). The 27-residue stretch of the amino terminus was predicted to
be the signal peptide containing a potential signal peptidase cleavage
site (16) between Ala124 and
Ala123.
Following the putative signal sequence and preceding the amino terminus of the mature protein, there is a pro-region of 123 residues. Finally, the sequence between the amino terminus, identified in the native protein, and the carboxyl terminus as indicated by the stop codon corresponds to a protein that encompasses 467 amino acids with a theoretical pI = 4.91 and a calculated molecular mass of 47,446 Da.
The S. maltophilia protease gene was termed StmPr1 (European Molecular Biology Laboratory accession number AJ291488); the term takes into account that another protease gene (StmPr2)2 was detected in S. maltophilia during preparation of this manuscript.
Comparison of the StmPr1 protease sequence with those of known bacterial serine proteases confirmed its relation to the subtilisin family of proteases (cf. Ref. 7). Alignment of the sequences indicated that Asp42, His105, and Ser289 form the putative catalytic triad characteristic for serine proteases. In the active site region, there is considerable homology with other subtilisins; in Fig. 3, conserved residues that are shared with subtilisin BPN' (17) and proteinase K (18) are marked with open boxes as typical representatives of the "classic" subtilisins. Nevertheless, the StmPr1 protease sequence reveals significant structural differences from these related enzymes due to large inserts adjacent to the catalytic His105 and Ser289. Therefore, compared with the catalytic triad formed by Asp32, His64, and Ser221 in the typical members of the subtilisin family, the architecture of the active site should be different in the StmPr1 protease. In addition, this enzyme has a longer carboxyl-terminal extension beyond the active site, which, together with the inserts in the catalytic region, makes the entire sequence almost 100 residues longer. With these structural properties, the StmPr1 protease is similar to the extracellular proteases of Xanthomonas campestris (19), D. nodosus (15), and Alteromonas sp. (20); the homology with these proteases is 49%, 40%, and 38% identity, respectively, for the mature proteins. On the other hand, there is only low homology within the region of the carboxyl-terminal extensions, and no homology can be seen between the pro-sequences. The sequence homology between the StmPr1 protease and the classic subtilisins is also lower, e.g. 23% identity with proteinase K (18) within the region of the mature enzymes.
Properties of the Enzyme
In view of the sequence differences between the well-characterized subtilisins and the StmPr1 protease, it was important to analyze the enzymatic activity of the new protease in detail.
The StmPr1 protease hydrolyzes the widely used chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA with a Km of 1.4 mM. This substrate was used for characterization of the enzyme purified from the native source.
Effect of pH
The enzyme activity of the purified StmPr1 protease was
measured in the pH range 4-11 (Fig.
4A) and showed a typical bell shape. The optimum pH was 9.0, classifying the StmPr1
protein as an alkaline protease. Pre-exposure of the protease to
extreme pH (0.1 M acetic acid, pH 3) for 10 min on ice
resulted in a 68% loss of enzyme activity.
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Modulation of Enzyme Activity A study of the salt requirement for enzyme activity was conducted by assaying the enzyme at pH 9. Raising the final NaCl concentration to 0.4 M increased activity about 4-fold (Fig. 4B). No further increase in enzyme activity was observed at higher salt concentrations (1 M NaCl was the maximum concentration tested). The stimulating effect of NaCl has also been reported for several other proteases of the subtilisin family (21). Enzyme activity was also found to be stimulated by calcium, which was effective at low concentrations: a maximum 3.5-fold increase in activity was observed at 50 mM calcium chloride. The effects of Na+ and Ca2+ were not additive. Ca2+ can be replaced by Mg2+ to achieve the same activating effect (data not shown). Thus, the StmPr1 protease is an enzyme dependent on bivalent metal ions; the strong activation effect of the cations can be explained by a conformational change leading to a catalytically more active conformation. Na+ possibly binds to the same site and may substitute for Ca2+ at higher concentrations.
A remarkable property of the StmPr1 protease is its relative stability toward the anionic detergent sodium dodecyl sulfate (Fig. 4C). The enzyme preserved 85% of its activity in the presence of 0.1% detergent, and even at a concentration of 1% dodecyl sulfate, 45% of proteolytic activity was retained. Similar results indicating a particular conformational stability have been reported for some, mainly microbial, proteases (cf. Ref. 22). By contrast, the mammalian serine protease chymotrypsin tested under the same conditions completely lost its activity at a concentration of 0.1% dodecyl sulfate.
To get more information on the type of protease, the effect of a series of protease inhibitors on StmPr1 enzyme activity was tested (Table I). The enzyme was effectively inhibited by antipain, chymostatin, and phenylmethylsulfonyl fluoride, whereas other serine protease inhibitors such as leupeptin, TLCK, and TPCK were not effective. The lack of inhibitory activity of TPCK is in contrast to the reported effect of this compound on subtilisin (21, 23). The StmPr1 protease is not inhibited by EDTA, presumably because the calcium bound to the enzyme cannot be chelated, and the protein remains structurally unaffected. This result demonstrates that metal ions are not directly involved in the catalytic mechanism, which is characteristic for subtilisins and other serine proteases.
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Of interest with respect to the pathogenic potential of the
StmPr1 protease was the observation that human plasma
protease inhibitors 1-antitrypsin and
2-macroglobulin could not abolish the proteolytic
activity of the enzyme; as shown below (Fig. 6), these two polypeptide
inhibitors themselves are subject to proteolytic digestion through the
bacterial protease.
The properties of the StmPr1 protease clearly show that this enzyme is different from proteases isolated from P. maltophilia in 1975 (24) and in 1985 (25). These enzymes are strongly inhibited by EDTA, whereas antipain, which is a potent inhibitor of the StmPr1 protease, was found to be ineffective (25). Moreover, both enzymes differ in molecular size from the protein reported here. Obviously, at that time, P. maltophilia was a heterogenous species due to other differentiation criteria applied. Therefore, these observations strongly suggest that the bacteria used at that time are not identical with the strain of S. maltophilia that served as a source of the StmPr1 protease.
Substrate Specificity In view of the pathogenic effect that the StmPr1 protease may exert in infected patients, the substrate specificity of this enzyme was studied in detail. This is a prerequisite for the development of specific inhibitors to be tested as therapeutic agents.
Proteolytic Activity toward the Oxidized Insulin B
Chain--
Proteolytic specificity of the StmPr1 protease was
determined using the oxidized insulin B chain as a substrate with a
known sequence. The proteolytic fragments were analyzed by HPLC and mass spectroscopy. A total of eight bonds were cleaved (Fig.
5). The results characterized the
protease as an endopeptidase with broad specificity. StmPr1
protease attacks peptide bonds comprising the carboxylic groups of both
hydrophobic and hydrophilic residues. Comparison with the
alkaline protease from D. nodosus and with subtilisin BPN'
showed that none of the specificity patterns is identical with that of
StmPr1 protease.
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P1-P4 Specificity-- P1 specificity of StmPr1 protease was investigated with a series of 10 tetrapeptide 4-nitroanilides in which only the amino acid residue in position P1 was varied (Table II). Determination of the specificity constant kcat/Km showed a strong preference for the positively charged side chain of lysine. The high efficiency of the enzyme is derived from both greater binding (lower Km) and increased turnover (higher kcat). The enzyme efficiently hydrolyzed substrates containing aromatic or aliphatic groups in position P1, but with a lower efficiency. The S1 subsite accepted the negatively charged side chains of glutamic and aspartic acid very poorly. The following order of specificity, characterized by the ratio kcat/Km, was observed.
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(Eq. 1) |
(i) The S1 subsite of StmPr1 protease is negatively charged. It can be supposed that a carboxylic group(s) is (are) located in this site. This can explain the high affinity for the side chain of lysine and the very low efficiency toward substrates with aspartic or glutamic acid in P1. S1 can also accommodate residues containing nonpolar side chains but with lower affinity.
(ii) Most probably, S1 is a deep and narrow "cavity" at the bottom of which negatively charged group(s) is (are) located. The best interaction is realized with the side chain of lysine (four methylene groups). Shortening of the chain by one CH2 group, as in the case of ornitine, drastically decreased the catalytic efficiency. The distorted binding of the bulky side chain of valine seems to be due to a steric repulsion and the narrow entrance of the cavity.
(iii) In general, the StmPr1 protease exhibits a mixed type of P1 specificity (trypsin-like and, to a lesser extent, subtilisin-like activity). This is unusual for subtilases.
The subsite S2 prefers Pro instead of Leu in position P2 (Table II). The low efficiency toward Suc-Phe-Leu-Phe-pNA is due to a decreased turnover number. Some subtilases, like Esperase, Savinase, and subtilisin BPN' exhibit an opposite preference (26, 27).
StmPr1 protease efficiently hydrolyzes tetrapeptide p-nitroanilides with different P3 residues. The enzyme definitely prefers Leu and Gly in position P3 (Table II). The absence of a considerable discrimination between the P3 residues with different nature can be explained by a location of the subsite S3 at or near to the surface of the protein globule. The following decreasing order of P3 specificity was observed: Leu > Gly > Phe = Ala > Glu. This order is completely different from those of other proteases of this family.
Subtilases exhibit a preference for the aromatic group of Phe in P4 because hydrophobic forces predominate in the S4-P4 interactions. As a result, Suc-Phe-Ala-Ala-Phe-Phe-pNA is one of the most favorable substrates for this group of proteases. However, the catalytic efficiency of the StmPr1 protease is 2 orders of magnitude lower than those of typical subtilases (Table II and Ref. 26). The low efficiency is due mainly to a decreased turnover number. This result again demonstrates the specific active site structure of the investigated protease, which is somewhat different from those of typical subtilases.
Reactivity toward Relevant Human Proteins
After having demonstrated with synthetic substrates the broad
specificity of StmPr1 protease, it was important to test
some human proteins that could be substrates in vivo. As
shown in Fig. 6, the enzyme degrades
protein components of connective tissue such as collagen and
fibronectin. This property of the bacterial protease may contribute to
the tissue destruction seen in infected patients. Also, the serum
component fibrinogen was completely degraded, indicating that the
StmPr1 protease may interfere with the process of blood
clotting. It has been shown above that the physiological protease
inhibitors 1-antitrypsin and
2-macroglobulin present in serum at high concentrations
are unable to abolish the StmPr1 proteolytic activity; Fig.
6, E and F, now demonstrates that these
protein inhibitors, too, are subject to degradation. It is noteworthy
that when immunoglobulin G was incubated with StmPr1
protease, the heavy chain appears to be cleaved at a specific site,
giving raise to two smaller fragments; Fig. 6F shows the result obtained with a mouse monoclonal IgG1. Polyclonal IgG from human
serum principally yielded the same result but with more diffuse bands
(data not shown) due to the heterogeneity of the immunoglobulin
fraction. Taken together, the StmPr1 protease appears to be
associated not only with tissue destruction but may also possess the
ability to inactivate components of the host defense mechanism.
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Cell-damaging Activity
To verify the biological significance of the obtained data,
cultures of human fibroblasts were exposed to supernatants of S. maltophilia (Fig. 7). After
application of the cell-free bacterial medium, significant changes in
cell morphology were observed: the cell layer partially condensed,
forming cell-free areas, and finally detached from the culture plate.
The same cell-damaging effect was achieved by addition of the purified
StmPr1 protein to the fibroblast culture (Fig.
7C). The destructive effect of both bacterial supernatant
and purified enzyme could be prevented by preincubation with
chymostatin, which has been shown above to be a potent inhibitor of the
protease. This experiment demonstrates that secretions of S. maltophilia are able to destroy living cells and that the StmPr1
protease is the major factor responsible for this effect. Therefore, it
seems likely that the tissue lesions seen in infected patients are a
consequence of StmPr1 action.
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Conclusions
A new bacterial protease with broad specificity has been characterized that is important from both a biochemical and a medical point of view. The sequence and the enzymic properties demonstrate that this protease is a new member of the family of subtilases. It differs from the classic subtilisins by its larger molecular size and, presumably, by the architecture of the catalytic site. In this respect, the StmPr1 protease is homologous to the extracellular proteases of X. campestris, a plant pathogen causing black rot in crucifers, and D. nodosus, which is the causative pathogen of ovine foot rot, a disease characterized by separation of the hoof from the epidermal tissue. In both cases, the pathological situation seems to be associated with proteolytic tissue damage. Consequently, the StmPr1 protease is likely to function as a pathogenic factor as well.
Broad spectrum antibiotic treatments causing bacterial selection
combined with the multiresistance of S. maltophilia force the development of new therapeutic strategies. A possible approach to
this problem is to interfere with the pathogenic mechanisms of the
bacteria (in the case of S. maltophilia, to suppress
protease-mediated tissue invasion and destruction). In this context,
inhibitors of the StmPr1 protease should be of therapeutic
value. It seems important, however, that such inhibitors do not affect
host proteases. Fortunately, little structural relationship seems to
exist between the prokaryotic and eukaryotic proteases, despite similar
mechanisms of action (cf. Ref. 8). Therefore, it should be
possible to design inhibitors with the required specificity. The
development of such discriminating inhibitors is not without
precedence: human immunodeficiency virus protease inhibitors, designed
on the basis of crystal structures of the target protein, have been
successfully introduced into therapy of AIDS. The data presented here
should pave the way toward determination of the StmPr1
protease structure. Crystallization of the protein will be facilitated
(cf. Ref. 28) by complexing with inhibitor molecules as
developed on the basis of the enzyme kinetics presented.
| |
ACKNOWLEDGEMENTS |
|---|
The skillful technical assistance of Sigrid Himpel is gratefully acknowledged. We thank Jan Tühscher for having performed the analysis of bacterial growth and protease production. We also thank Dr. David Sugar for critical reading of the manuscript.
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FOOTNOTES |
|---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ291488.
¶ Present address: Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Present address: Bernhard-Nocht-Institut für
Tropenmedizin, D-20359 Hamburg, Germany.
** To whom correspondence should be addressed: Institut für Medizinische Biochemie und Molekularbiologie, Universitätsklinikum Hamburg-Eppendorf Martinstrasse 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-4459; Fax: 49-40-42803-6818; E-mail: weber@uke.uni-hamburg.de.
Published, JBC Papers in Press, January 16, 2002, DOI 10.1074/jbc.M109525200
2 S. Windhorst and W. Weber, manuscript in preparation.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
HPLC, high pressure
liquid chromatography;
TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone;
TPCK, L-1- tosylamido-2-phenylethyl
chloromethyl ketone.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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), phosphate (
), or ethylendiamine (
