From the Department of Microbiology,
Immunology and Parasitology, Louisiana State University Health Sciences
Center and § Louisiana State University Eye Center, New
Orleans, Louisiana 70112
Received for publication, September 3, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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Protease IV is a lysine-specific endoprotease
produced by Pseudomonas aeruginosa whose activity has been
correlated with corneal virulence. Comparison of the protease IV amino
acid sequence to other bacterial proteases suggested that amino acids
His-72, Asp-122, and Ser-198 could form a catalytic triad that is
critical for protease IV activity. To test this possibility,
site-directed mutations by alanine substitution were introduced into
six selected residues including the predicted triad and identical
residues located close to the triad. Mutations at any of the amino
acids of the predicted catalytic triad or Ser-197 caused a loss of
enzymatic activity and absence of the mature form of protease IV. In
contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that
accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the
lysine-specific endoprotease of Achromobacter lyticus
suggested three possible disulfide bonds in protease IV. These results
identify the catalytic triad of protease IV, demonstrate that
autodigestion is essential for the processing of protease IV into a
mature protease, and predict sites essential to enzyme conformation.
Pseudomonas aeruginosa is a major opportunistic
pathogen that is often associated with burns, cancers, cystic fibrosis,
and immune deficiency diseases (1-6). P. aeruginosa is also
well recognized as the most important Gram-negative bacterium causing severe keratitis, especially in contact lens wearers (7-9). This keratitis progresses rapidly and may result in permanent loss of vision
(10, 11). The ability of P. aeruginosa to produce several
extracellular enzymes, such as elastase (LasB, pseudolysin), alkaline
protease (aeruginolysin), LasA (staphylolysin), and LasD (staphylolysin), is considered to be important in its pathogenicity. Protease IV (lysyl endopeptidase; EC 3.4.24.26) has been demonstrated to correlate with corneal virulence. Protease IV has also recently been
identified as the iron-regulated protein PrpL (12). Protease IV is a
lysine-specific protease with molecular mass of 26 kDa that has been
identified in culture supernatants of P. aeruginosa (13-15). Protease IV was first characterized in P. aeruginosa strain PA103-29, a strain reported to be deficient in
alkaline protease, elastase A, and elastase B production (16).
Recently, the nucleotide sequence of the protease IV gene from P. aeruginosa PA103-29 was determined and found to correspond to a
sequence between base pairs 4,671,318 and 4,672,706 of the P. aeruginosa PAO1 chromosome (The Pseudomonas Genome
Project identifies this gene as PA4175) (1). The protease IV gene is
located in a 1388-bp open reading frame, encoding a protein of 463 amino acids (17). Similar to elastase B, this protein consists of three
domains: signal sequence, propeptide domain, and mature protease.
Protease IV is synthesized as a pre-proenzyme that is processed
intracellularly and secreted into the extracellular milieu as the
mature protease (33).
The 26-kDa mature protease is a unique extracellular enzyme that
specifically cleaves substrates on the carboxyl side of lysine residues
as determined by substrate susceptibility tests. Protease IV is also
capable of degrading a variety of host defense proteins including
fibrinogen, plasminogen, immunoglobulin G, and the complement proteins
C1q and C3. The enzyme can be completely inhibited by a protease
inhibitor, tosyl-lysyl-chloromethylketone (13). Protease IV has
been implicated as a virulence factor that contributes to the
pathogenicity of Pseudomonas keratitis (13, 14, 18). Purified protease IV (50-200 ng) induced corneal epithelial damage within 3 h after injection into the corneal stroma and increased the virulence of protease IV-deficient bacteria (19). Protease IV may
be involved in the processing mechanism of elastase A (LasA), because
it has been reported that a lysine-specific protease of P. aeruginosa converts the LasA proenzyme to the active enzyme (20).
The protease IV gene has been cloned and expressed in Pseudomonas
putida providing a more abundant supply of protease than that
obtained from the naturally expressed chromosomal P. aeruginosa gene (33). Expression of protease IV in P. putida revealed a protein of 48 kDa containing the three domains
of protease IV: a signal sequence, a propeptide, and a mature protease.
Also detected was a 45-kDa protein (proenzyme) that contained the
propeptide and mature protease domains but not the signal peptide. In
an experimental model of keratitis, expression of protease IV by P. putida caused a 3-fold increase in the clinical score of
infected eyes relative to eyes infected with P. putida
lacking the protease IV gene (33).
The present study was undertaken to study the sequence of protease IV
relative to other bacterial proteases and to determine the amino acid
residues of protease IV responsible for the enzymatic activity.
Included in this study is the synthesis and testing of multiple
site-directed mutants involving residues proposed to comprise the
catalytic site of protease IV.
Bacterial Strains, Plasmids, and Growth
Conditions--
Escherichia coli strain DH5 Site-directed Mutagenesis--
Site-directed mutagenesis was
accomplished by the overlap extension method using PCR (22). The
protease IV gene was obtained by amplifying the structural gene from
the chromosome of P. aeruginosa strain PA103-29 using the
GC-rich PCR system (Roche Molecular Biochemicals) under the following
conditions: 3 min at 95 °C, followed by 30 cycles of 30 s at
95 °C for denaturing, 30 s at 60 °C for annealing, 2 min at
72 °C for extension and ending with an incubation at 72 °C for 7 min. For generating mutations in the protease IV gene, two pairs of
primers were used to direct the synthesis of two fragments, each
inclusive of an overlapping region containing a replacement of His-72,
His-116, Asp-122, Ser-197, Ser-198, or Ser-200 with an Ala
residue (Table I). The two PCR products, with an overlap of 27 base pairs at one end of each fragment,
were then combined for the second round of PCR designed to amplify the
entire protease IV gene. Oligonucleotide primers were designed to
amplify the protease IV gene with recognition sites for the restriction
enzyme EcoRI at the locus coding for the N-terminal amino
acids and the restriction enzyme BamHI site at the locus
coding for the C-terminal amino acids. A 1388-base pair fragment of the
PCR products was ligated into a PCR cloning vector (TOPO TA cloning;
Invitrogen) and transformed into chemically competent TOP10 E. coli as described by the manufacturer. Plasmid DNA was purified
from transformants, and the protease IV gene was excised from the
cloning vector by EcoRI/BamHI restriction digestion. The EcoRI/BamHI DNA fragment
containing the protease IV gene was subcloned into pUCP20 and
transformed into electrocompetent E. coli DH5
Purification of plasmid DNA was performed by the alkali lysis method
using a QIAprep Spin Miniprep kit (Qiagen Inc., Valencia, CA).
Large-scale plasmid preparations were performed by Qiagen Plasmid Midi
kit (Qiagen). Restriction enzymes and T4 DNA ligase were purchased from
New England Biolabs, Inc. (Beverly, MA).
Purification of Protease IV--
For purification of the
intracellular wild-type and mutated protease IV, plasmid-containing
E. coli was grown at 37 °C in 2 liters of LB medium.
Cells were pelleted, washed three times, and resuspended in 50 ml of 10 mM Tris-HCl, pH 7.0. Cells were ruptured by sonication
(Branson Sonifier 250; Branson Ultrasonics Corp., Danbury, CT). Intact
cells were removed by centrifugation at 8,000 × g for
20 min at 4 °C. The extracts were dialyzed overnight against 10 mM ammonium acetate buffer, pH 6.4. The dialyzed
supernatant was applied to a CM cation exchange matrix (Bio-Rad,
Hercules, CA) and was washed with 10 mM ammonium acetate
buffer, pH 6.4. A pH gradient, generated by mixing 10 mM
ammonium acetate buffer at pH 9.0 with 10 mM ammonium
acetate buffer at pH 6.4, was used to elute the protein. The eluted
fractions containing protease IV were assayed by Western blotting with
rabbit polyclonal antiserum prepared against a recombinant form of
mature protease IV (33). The reactive fractions were pooled and then
concentrated to 1.0 ml using a stirred ultrafiltration cell with a
10-kDa cutoff filter membrane (YM10; Amicon Inc., Beverly, MA) and
loaded onto a Sephacryl S-300 molecular sieve matrix (Amersham
Biosciences). The fractions were eluted with 10 mM
Tris-HCl, pH 7.0, and assayed by immunoblotting. Positive fractions
were pooled and concentrated to 1.0 ml. Total protein was determined
using the bicinchoninic acid assay (Sigma). The purity of the sample
was determined by 12% SDS-PAGE and visualized by silver staining
(Bio-Rad). The purification of extracellular protease IV was
performed using the method of Engel et al. (13), as modified
by Traidej et al. (33).
Colorimetric Substrate Assay for Protease IV
Activity--
Protease IV activity was determined by the hydrolysis of
the chromogenic substrate, Chromozym PL
(tosyl-Gly-Pro-Lys-p-nitroanilide; Sigma), as described by
O'Callaghan et al. (14). End point reactions were incubated
at 37 °C for 30 min or as stated in each experiment. Kinetic
analysis was performed at 25 °C by reading the optical density every
2 min for 30 min to measure the increase in optical density per min
( SDS-PAGE, Immunoblotting, and N-terminal Sequence
Analysis--
Proteins were separated by 12% SDS-PAGE under reducing
conditions and electroblotted by standard laboratory methods (23). Rabbit polyclonal antiserum prepared against a recombinant form of
mature protease IV was the primary antibody, and detection of immune
complexes was done with ECL Western blotting detection reagents
(Amersham Biosciences).
For N-terminal sequence analysis, proteins were electrotransferred from
SDS-polyacrylamide gel to Sequi-blot polyvinylidene difluoride membrane
(Bio-Rad). N-terminal amino acids were determined by Core Laboratories,
Louisiana State University Health Sciences Center, New Orleans, LA.
Comparison of Amino Acid Sequences of Pseudomonas Protease IV with
Other Serine Proteases--
The amino acid sequence of mature protease
IV was subjected to a Blastp search against the Microbial Genomes
database (www.ncbi.nlm.nih.gov/BLAST/) for related proteins. The
three proteases found to be related to protease IV were endoproteinase
Arg-C (44% identity) of Lysobacter enzymogenes, lysyl
endopeptidase (31% identity) of L. enzymogenes, and
Achromobacter protease I (30% identity) of
Achromobacter lyticus (Fig. 1). These three proteins are
classified as serine proteases and, like protease IV, cleave substrates
containing a lysine residue (12, 23-26). Both protease IV and these
related proteases are synthesized as proenzymes that are subsequently
processed to mature extracellular forms (24, 27, 29).
The active site of Achromobacter protease I, like that of
trypsin and chymotrypsin (30), is described as a catalytic triad consisting of histidine, aspartic acid, and serine (31). The active
site sequences of these proteases are similar to sequences in the
mature protease IV. Based on these sequence similarities, the catalytic
triad of protease IV is suggested to be comprised of His-72, Asp-122,
and Ser-198. In addition, six cysteines of protease IV are also aligned
with those of the other related proteases. Based on similarities to the
A. lyticus protease I positioning of disulfide bonds, the
disulfide bonds present in protease IV are anticipated to align
as indicated in Fig. 1.
Effect of Active Site Mutations on Enzymatic Activity--
To
analyze the protease IV enzyme in terms of the amino acid residues
comprising its active site, site-directed mutations were introduced
into cloned copies of the protease IV gene for each of the following
amino acids: His-72, His-116, Asp-122, Ser-197, Ser-198, and Ser-200.
The amino acids (histidine and serine, as well as aspartic acid) were
substituted with alanine to eliminate functional groups from the enzyme
at the selected positions. Successful mutation of the protease IV gene
at each chosen site was verified by DNA sequencing (data not shown).
To examine the effect of active site mutations on the enzymatic
activity of protease IV, culture supernatants of E. coli
expressing wild-type and mutated forms of protease IV were analyzed for
their ability to hydrolyze a lysine-containing substrate, Chromozym PL.
E. coli expressing wild-type protease IV possessed high
proteolytic activity as demonstrated by chromogenic substrate cleavage
(Table II). Mutants with protease IV
sequence substitutions of alanine at residues His-116 or Ser-200 showed
similar protease activity comparable with that of E. coli
expressing wild-type protease IV. In contrast, mutants with an alanine
substitution at His-72, Asp-122, Ser-197, or Ser-198 displayed an
insignificant amount of proteolytic activity. These findings suggest
that the proposed catalytic triad constituents (His-72, Asp-122, and
Ser-198), as well as Ser-197, are essential for the proteolytic
activity of protease IV.
Effect of Active Site Mutations on the Protease IV Processing
Mechanism--
To determine the effect of alanine substitutions on
protease IV processing, the presence of wild-type and mutated protease IV gene products in cell lysates was examined by immunoblotting using
rabbit anti-mature protease IV antibodies. For the wild-type protease
IV gene expressed in E. coli, the findings were similar to
those detected previously for protease IV expression in P. putida; that is, the mature protease (26 kDa), as well as
intracellular proteins of 48 and 45 kDa, were present (Fig.
2) (33). The 48-kDa protein has been
reported to be the full-length protease IV gene product
(pre-proenzyme), and the 45-kDa protein is a proenzyme inclusive of the
propeptide and the 26-kDa mature protease domains but lacking the
signal sequence (33). Unlike the wild-type protease IV, each mutated
protease IV gene product with an alanine substitution at an amino acid
proposed to be part of the catalytic triad (His-72, Asp-122, or
Ser-198) was not processed intracellularly to the 26-kDa mature form
but instead remained in the 48-kDa pre-proenzyme and 45-kDa proenzyme
forms. This finding is consistent with the possibility that the 45-kDa
proenzyme form of wild-type protease IV undergoes autoprocessing to
separate the propeptide and the mature protease domains.
The mutation at Ser-197, the residue located next to Ser-198 of the
proposed catalytic triad, also caused an accumulation of the 48-kDa
pre-proenzyme and 45-kDa proenzyme forms. However, mutations at either
His-116 or Ser-200 did not result in an accumulation of the 48-kDa
pre-proenzyme or 45-kDa proenzyme forms and did allow the processing of
the protein to the mature protease form of 26 kDa. These results
indicate that His-72, Asp-122, Ser-197, and Ser-198 of mature protease
IV, but not His-116 or Ser-200, are amino acid residues that are not
only essential for proteolytic activation of protease IV but also play
a crucial role in autoprocessing of the proenzyme.
Characterization of Alanine-substituted Protease IV--
To
further characterize the alanine-substituted protease IV mutations,
E. coli carrying pH72, which lacked protease activity, and
E. coli harboring pH116, whose protease activity was
retained, were used as sources for purification of intracellular
protease IV. E. coli carrying pH72 contained a protein of 45 kDa whereas cells carrying pH116 contained a protein of 26 kDa (Fig.
3). The 26-kDa protein of pH116 had an
N-terminal amino acid sequence of AGYRDGFGAS, which was identical to
that of the mature protease IV of P. aeruginosa.
Furthermore, the N-terminal amino acid sequence obtained from the
purified 45-kDa protein of pH72 was APGASE, matching the amino acid
sequence of protease IV beginning at position 25 of the full-length
protease IV. The APGASE sequence suggests that the cleavage site for
signal peptidase is located between amino acid residues 24 and 25. The
purified proteins of 45 and 26 kDa were analyzed for protease activity
by the Chromozym PL assay, and only the 26-kDa mature protease was
enzymatically active (data not shown).
In Vitro Processing of Protease IV--
There was a possibility
that mutations at His-72, Asp-122, Ser-197, or Ser-198 caused a
conformational change in the proenzyme form of protease IV that
rendered it resistant to proteolytic processing from the proenzyme form
to the mature 26-kDa form. To test this possibility, the 45-kDa
proenzyme of the His-72 mutant was purified and then incubated in
vitro for 1 h with the 26-kDa mature protease IV (0.1 µg)
purified from P. aeruginosa PA103-29 culture supernatants.
The samples were analyzed by immunoblotting, and the proteolytic
activity was determined by using the Chromozym PL substrate.
Incubation of the His-72 proenzyme alone did not induce protein
processing into the mature protease form as evidenced by the 45-kDa
band that was present after incubation
(Fig. 4). Incubation of the His-72
proenzyme in the presence of active protease IV resulted in the
disappearance of the 45-kDa protein. Furthermore, incubation of the
His-72 proenzyme with heat-inactivated protease IV failed to cause the
disappearance of the His-72 45-kDa protein. The liberation of the
His-72 mature protease domain (with alanine substituted for histidine
at position 72) caused no increase in the total protease IV activity
(data not shown). These results on the His-72 mutant suggest that the
45-kDa proenzyme with an alanine substitution was not resistant to the
proteolytic cleavage that naturally occurs in the production of the
mature protease IV domain. The ability of this proenzyme to be cleaved
into the 26-kDa protein lacking proteolytic activity is consistent with the proposal that the mutated amino acid is part of the active site of
protease IV.
This study determined sequence similarities between protease IV
and other bacterial proteases specific for lysine residues and, based
on sequence similarities, predicted three amino acids, histidine 72, aspartic acid 122, and serine 198, that likely comprise the catalytic
triad of protease IV. This prediction was supported by site-directed
mutagenesis that demonstrated a dramatic loss of enzymatic activity
resulting from mutation in any of these three residues. These results
also revealed that the junction of amino acids 24-25 is the site of
cleavage by a signal peptidase that removes the signal sequence from
the full-length pre-proenzyme, leaving the proenzyme intact. The
results also demonstrate that protease IV undergoes autodigestion to
liberate the mature protease from the 45-kDa proenzyme containing both
the propeptide and mature protease domains.
The protease IV gene mutated at any of the proposed active site amino
acids produced both the pre-proenzyme (48 kDa) and the proenzyme (45 kDa) that were not processed to the mature protease (26 kDa).
Pre-proenzyme and proenzyme were produced in cells with the wild-type
protease IV gene, but these native proteins, unlike the mutant
proteins, were processed naturally into the enzymatically active
protease IV. The failure of the mutated proenzymes to be processed into
mature proteases is apparently because of their loss of the functional
catalytic site and not to the inaccessibility of the lysine cleavage
site to the mature protease IV. Protease IV purified from P. aeruginosa appeared to be capable of digesting the mutated
proenzyme into a 26-kDa protein that, unlike wild-type mature protease,
lacked enzyme activity because of its alanine substitution at position
72. These results demonstrate that a mutation at a single amino acid
needed for the proposed catalytic site did not cause the proenzyme to
misfold into an unfavorable conformation for proteolytic cleavage at
the position between the propeptide and mature protease domains. These
findings infer that the natural separation of the propeptide from the
mature protease is a result of autoprocessing involving the cleavage of
the 45-kDa proenzyme at the lysine residue present at the junction between the propeptide and the mature protease domains. The significant role of the proposed catalytic triad of protease IV in proteolytic activity is in agreement with findings for related serine proteases (24, 30, 31).
In addition to the catalytic triad, serine at position 197 of protease
IV also showed a crucial role in processing and enzyme activity. This
amino acid is located adjacent to Ser-198, a constituent of the
proposed catalytic triad. To date, the three-dimensional structure of
protease IV has not been resolved. Our prediction that the catalytic
triad of protease IV consisting of His-72, Asp-122, and Ser-198 was
originally based on an alignment of the primary structure of protease
IV with other lysine-specific endoproteases. A possibility is that
Ser-197 could be a constituent of the catalytic triad instead of
Ser-198. However, when the amino acid sequence of mature protease IV
was compared with the mammalian serine proteases (trypsin and
chymotrypsin), Ser-197 and Ser-198 of protease IV aligned with Asp-194
and Ser-195 of trypsin and chymotrypsin, respectively. X-ray
crystallography revealed that Asp-194 of trypsin has an important role
in the structural conformation whereas Ser-195 was part of the
catalytic triad of trypsin and chymotrypsin (28, 32). The Asp-194
residue forms a salt bridge with Ile-16 near the N terminus that helps
establish the proper conformation for the enzymatic activity of trypsin
and chymotrypsin. Ser-197 of protease IV, like Asp-194 of trypsin and
chymotrypsin, could be responsible for the proper folding of protease
IV. Therefore, an amino acid substitution at Ser-197 could result in a
structural change that would alter the folding of the tertiary
structure of protease IV into the correct active form and could prevent enzymatic activity.
Based on the similarity between protease IV and other bacterial serine
proteases, speculation can be offered regarding the disulfide bonds in
protease IV. The positions of all six cysteines of protease IV are
aligned to cysteines in the amino acid sequences of other related
proteases. Critical to the structure of Achromobacter protease I is the disulfide bond that forms between the most distant cysteines (i.e. Cys-6 and Cys-216) (24). Protease IV has
cysteines in similar positions (Cys-13 and Cys-220) that could function to stabilize protease IV in an enzymatically active form. This possibility is consistent with the sensitivity of protease IV activity
to the reducing agent, In summary, we have determined the essential amino acid residues of
protease IV that affect the catalytic activity. The processing of
protease IV from a 45-kDa proenzyme to the secreted mature protease IV
was dependent upon the catalytic activity of protease IV, demonstrating
that protease IV undergoes autoprocessing in the P. putida
system presented here. The processing of protease IV could be augmented
by other proteases in a P. aeruginosa system. We have also
identified the cleavage site for the signal peptidase that converts the
48-kDa pre-proenzyme to the 45-kDa proenzyme. The determination of the
three-dimensional structure of protease IV is still needed to gain a
better understanding of the structure/function relationship of protease IV.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was grown at
37 °C in LB broth (1.0% tryptone, 0.5% yeast extract, and 1.0%
NaCl, pH 7.0). When appropriate, carbenicillin (100 µg/ml) was
incorporated into the medium for plasmid selection. P. aeruginosa strain PA103-29 was cultivated in tryptic soy broth
(Difco, Detroit, MI) or M9 minimal medium containing 50 mM
monosodium glutamate, 1 mM MgSO4, and 1%
glycerol. Growth of bacterial cultures was performed at 37 °C as
described previously (13). E. coli strain DH5
was
purchased from Invitrogen. P. aeruginosa strain
PA103-29 was kindly provided by Dr. P. V. Phibbs, East Carolina
University, Greenville, NC. Strain PA103-29 was originally described by
Dr. D. E. Ohman (16). The plasmid pUCP20 was obtained from Dr.
J. A. Hobden, Wayne State University, Detroit, MI and originally
described by Dr. S. E. West (21).
. The
resulting plasmids with the protease IV gene and with a replacement of
His-72, His-116, Asp-122, Ser-197, Ser-198, or Ser-200 were designated
pPIV, pH72, pH116, pD122, pS197, pS198, and pS200, respectively. These
plasmids were sequenced to verify the presence of correct
mutations.
Oligonucleotide primers used in PCR and DNA sequencing
A/min). One unit of activity was defined as the amount
of enzyme that caused an optical density increase at 410 nm of 1 A/min under the assay conditions.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
Alignment of the amino acid sequence of the
mature protease IV of P. aeruginosa with other serine
proteases. Amino acids identical to Pseudomonas
protease IV (PIV) are boxed and shaded
lightly. His-72, Asp-122, and Ser-198 of protease IV,
corresponding to His, Asp, and Ser of other serine proteases, are
proposed as components of the protease IV catalytic triad and are in
bold and in boxes. The six cysteines are in bold
and in dotted boxes. The cysteines for possible sites of
three disulfide bonds are labeled with *, , or
above
each of the paired cysteine residues. Arg-C is the
designation for endoproteinase Arg-C of L. enzymogenes,
Lys is for lysyl endopeptidase of L. enzymogenes,
and AP-1 is for Achromobacter protease I of
A. lyticus.
Proteolytic activity of site-directed mutants of protease IV
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Fig. 2.
Detection of wild-type and mutated protease
IV gene product expressed in transformed E. coli.
Proteins in cell extracts were subjected to SDS-PAGE under reducing
conditions and immunoblotted with rabbit polyclonal antisera directed
against mature protease IV. Immune complexes were visualized with a
protein A conjugate. Lane 1, E. coli/pUCP20;
lane 2, E. coli/pPIV; lane 3, E. coli/pH72; lane 4, E. coli/pH116; lane
5, E. coli/pD122; lane 6, E. coli/pS197; lane 7, E. coli/pS198;
lane 8, E. coli/pS200; and lane 9,
purified protease IV from PA103-29. P1, pre-proenzyme,
48-kDa full-length protease IV gene product; P2, 45-kDa
proenzyme; M, 26-kDa mature protease IV.
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Fig. 3.
SDS-PAGE analysis of the purified mutated
forms of protease IV. The mutated forms of protease IV at His-72
and His-116 were purified by ion exchange and gel filtration
chromatography. A 1-µg aliquot of protein was subjected to SDS-PAGE.
Protein bands were visualized by Coomassie staining. Lane 1,
purified 45-kDa proenzyme from E. coli/pH72; lane
2, purified 26-kDa mature protein from E. coli/pH116;
and lane 3, purified mature protease IV from P. aeruginosa PA103-29.
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Fig. 4.
In Vitro processing of mutant
proenzymes by mature protease IV. A 0.5-µg aliquot of the
purified protease IV proenzyme (45 kDa) from E. coli/pH72, a
mutant with an alanine substitution at His-72, was incubated at
42 °C for 1 h with either 0.1 µg of the purified 26-kDa
mature protease IV from P. aeruginosa PA103-29 or with
heat-inactivated mature protease IV. The mixtures underwent SDS-PAGE
and subsequent immunoblotting. Lane 1, purified 45-kDa
His-72 proenzyme from E. coli/pH72; lane 2,
mature protease IV of P. aeruginosa PA103-29; lane
3, purified 45-kDa His-72 proenzyme incubated with active protease
IV; lane 4, heat-inactivated protease IV of P. aeruginosa PA103-29; and lane 5, purified 45-kDa His-72
proenzyme and heat-inactivated protease IV.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-mercaptoethanol (13).
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ACKNOWLEDGEMENTS |
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We thank Dr. Iris Lindberg and Joseph Dajcs for critical reading of the manuscript and Julian Reed for technical assistance.
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FOOTNOTES |
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* This work was supported by Grant EYI 12961 from NEI, National Institutes of Health.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) AY062882.
¶ To whom correspondence should be addressed: Dept. of Microbiology, Immunology and Parasitology, LSU Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-4072; Fax: 504-568-2918; E-mail: rocall@lsuhsc.edu.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M208973200
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1. | Bodey, G. P., Bolivar, R., Fainstein, V., and Jadeja, L. (1983) Rev. Infect. Dis. 5, 27 |
2. | Bodey, G. P. (2001) Curr. Opin. Infect. Dis. 14, 403-407[Medline] [Order article via Infotrieve] |
3. | Koch, C., and Hoiby, N. (2000) Respiration 67, 239-247[CrossRef][Medline] [Order article via Infotrieve] |
4. | Koch, C., and Hoiby, N. (1993) Lancet 341, 1065-1069[Medline] [Order article via Infotrieve] |
5. | Mendelson, M. H., Gurtman, A., Szabo, S., Neibart, E., Meyers, B. R., Policar, M., Cheung, T. W., Lillienfeld, D., Hammer, G., and Reddy, S. (1994) Clin. Infect. Dis. 18, 886-895[Medline] [Order article via Infotrieve] |
6. | Doring, G. (1997) Monaldi. Arch. Chest Dis. 52, 363-366[Medline] [Order article via Infotrieve] |
7. | Alfonso, E., Mandelbaum, S., Fox, M. J., and Forster, R. K. (1986) Am. J. Ophthalmol. 101, 429-433[Medline] [Order article via Infotrieve] |
8. | Sweeney, D. F., Stapleton, F., Leitch, C., Taylor, J., Holden, B. A., and Willcox, M. D. (2001) Optometry Vis. Sci. 78, 100-105 |
9. | Stapleton, F., Dart, J. K., Seal, D. V., and Matheson, M. (1995) Epidemiol. Infect. 114, 395-402[Medline] [Order article via Infotrieve] |
10. | Arffa, R. C., and Eve, F. R. (1991) Int. Ophthalmol. Clin. 31, 89-110[Medline] [Order article via Infotrieve] |
11. | Dart, J. K. (1988) Br. J. Ophthalmol. 72, 926-930[Abstract] |
12. |
Wilderman, P. J.,
Vasil, A. I.,
Johnson, Z.,
Wilson, M. J.,
Cunliffe, H. E.,
Lamont, I. L.,
and Vasil, M. L.
(2001)
Infect. Immunity
69,
5385-5394 |
13. |
Engel, L. S.,
Hill, J. M.,
Caballero, A. R.,
Green, L. C.,
and O'Callaghan, R. J.
(1998)
J. Biol. Chem.
273,
16792-16797 |
14. | O'Callaghan, R. J., Engel, L. S., Hobden, J. A., Callegan, M. C., Green, L. C., and Hill, J. M. (1996) Invest. Ophthalmol. Vis. Sci. 37, 534-543[Abstract] |
15. | Toder, D. S., and Gambello, M. J. (1992) Abstracts of ASM Meeting, New Orleans, LA, May, 1992 , p. 104, American Society for Microbiology, Washington, D. C. |
16. | Ohman, D. E., Cryz, S. J., and Iglewski, B. H. (1980) J. Bacteriol. 142, 836-842[Medline] [Order article via Infotrieve] |
17. | Caballero, A. R., Thibodeaux, B. A., Marquart, M. E., Moreau, J. M., Traidej, M., and O'Callaghan, R. J. (2001) Invest. Ophthalmol. Vis. Sci. 42, S739 |
18. | Engel, L. S., Hobden, J. A., Moreau, J. M., Callegan, M. C., Hill, J. M., and O'Callaghan, R. J. (1997) Invest. Ophthalmol. Vis. Sci. 38, 1535-1542[Abstract] |
19. | Engel, L. S., Hill, J. M., Moreau, J. M., Green, L. C., Hobden, J. A., and O'Callaghan, R. J. (1998) Invest. Ophthalmol. Vis. Sci. 39, 662-665[Abstract] |
20. |
Kessler, E.,
Safrin, M.,
Gustin, J. K.,
and Ohman, D. E.
(1998)
J. Biol. Chem.
273,
30225-30331 |
21. | West, S. E. H., Schweizer, H. P., Dall, C., Sample, A. K., and Runyen-Janexky, L. J. (1994) Gene 128, 81-86 |
22. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
24. |
Tsunasawa, S.,
Masaki, T.,
Hirose, M.,
Soejima, M.,
and Sakiyama, F.
(1989)
J. Biol. Chem.
264,
3832-3839 |
25. | Masaki, T., Tanabe, M., Nakamura, K., and Soejima, M. (1981) Biochim. Biophys. Acta 660, 44-50[Medline] [Order article via Infotrieve] |
26. | Jekel, P. A., Weijer, W. J., and Beintema, J. J. (1983) Anal. Biochem. 134, 347-354[Medline] [Order article via Infotrieve] |
27. | Wright, D. S., Graham, L. D., and Jennings, P. A. (1998) Biochim. Biophys. Acta 1443, 369-374[Medline] [Order article via Infotrieve] |
28. | Birktoft, J. J., and Blow, D. M. (1972) J. Mol. Biol. 68, 187-240[Medline] [Order article via Infotrieve] |
29. | Li, S. L., Norioka, S., and Sakiyama, F. (1990) J. Bacteriol. 172, 6506-6511[Medline] [Order article via Infotrieve] |
30. | Dodson, G., and Wlodawer, A. (1998) Trends Biochem. Sci. 23, 347-352[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Norioka, S.,
Ohta, S.,
Ohara, T.,
Lim, S.,
and Sakiyama, F.
(1994)
J. Biol. Chem.
269,
17025-17029 |
32. | Stroud, R. M., Kay, L. M., and Dickerson, R. E. (1974) J. Mol. Biol. 83, 185-208[Medline] [Order article via Infotrieve] |
33. | Traidej, M., Caballero, A. R., Marquart, M. E., Thibodeaux, B. A., and O'Callaghan, R. J. (2002) Invest. Ophthalmol. Vis. Sci., in press |