From the Institute of Molecular Biology and
§ Faculty of Chemistry and Regional Laboratory, Jagiellonian
University, 31-120 Kraków, Poland and the ¶ Department of
Biochemistry and Molecular Biology, University of Georgia,
Athens, Georgia 30602
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ABSTRACT |
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Porphyromonas gingivalis possesses a
complex proteolytic system, which is essential for both its growth and
evasion of host defense mechanisms. In this report we characterized,
both at a protein and genomic level, a novel peptidase of this system
with prolyl tripeptidyl peptidase activity. The enzyme was purified to
homogeneity, and its enzymatic activity and biochemical properties were
investigated. The amino acid sequence at the amino terminus and of
internal peptide fragments enabled identification of the gene encoding
this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl
oligopeptidase family of serine proteases. However, it does not share
any structural similarity to other tripeptidyl peptidases, which belong
to the subtilisin family. The production of prolyl tripeptidyl
peptidase may contribute to the pathogenesis of periodontal tissue
destruction through the mutual interaction of this enzyme, host and
bacterial collagenases, and dipeptidyl peptidases in the degradation of
collagen during the course of infection.
Porphyromonas gingivalis, a Gram-negative,
asaccharolytic, anaerobic bacterium, is one of the predominant members
of the human periodontal flora and has been strongly implicated as a
causative agent of adult type periodontitis (1, 2). At least part of
its pathogenic potential appears to be attributable to the production
of a variety of proteolytic enzymes that facilitate, directly or
indirectly, both local tissue damage and evasion of host defense
mechanisms (for review, see Ref. 3). Recent studies have indicated that
this periodontopathogen produces at least seven different enzymes
belonging to the cysteine and serine catalytic classes of peptidases,
among which three cysteine proteinases (gingipains) are predominant
(for review, see Ref. 4). Because their structure, function, enzymatic
properties, and pathological significance are known, these
endopeptidases are the best characterized group of P. gingivalis enzymes. From in vitro studies it is
apparent that two gingipain Rs (HRgpA and RgpB), enzymes specific for
cleavage at Arg-Xaa peptide bonds, have a significant potential to
contribute to the development and/or maintenance of a pathological
inflammatory state in infected periodontal pockets through (i)
activation of the kallikrein-kinin cascade (5, 6), (ii) the release of neutrophil chemotactic activity from native and oxidized C5 of the
complement pathway (7), and (iii) activation of both factor X (8) and
prothrombin.1 This latter
effect leads to the uncontrolled generation of thrombin, an enzyme with
multiple functions, including strong proinflammatory activity (9). In
addition, gingipain K, an enzyme that cleaves Lys-Xaa peptide bonds,
degrades fibrinogen, and this may add to a bleeding on probing tendency
associated with periodontitis (10). Finally, the presence of a
hemagglutinin adhesion domain in the noncovalent multiprotein complexes
of HRgpA and gingipain K (11) suggests participation of these enzymes
in the binding of P. gingivalis to extracellular matrix
proteins, which may facilitate tissue invasion by this pathogen.
In comparison with the gingipains, relatively little is known about
other cysteine proteinases produced by P. gingivalis. Two
genes, referred to as tpr (12) and prtT (13),
have been cloned and sequenced, and although they encode putative
papain- and streptopain-like cysteine proteinases, respectively,
neither has been purified and characterized at the protein level.
However, a gene homologous to prtT has now been identified
in the partially sequenced P. gingivalis genome, and the
proteinase encoded by this gene, referred to as periodontain, has been
purified from culture media and
characterized.2
Despite the fact that the presence of serine proteinase activity in
cultures of P. gingivalis has been known for several years (14), only limited information is available about such enzymes. Indeed,
a serine endopeptidase has been isolated from culture media, although
it was only superficially characterized (15). On the other hand, an
enzyme referred to as glycylprolyl peptidase (dipeptidyl peptidase IV)
was found to be associated with bacterial surfaces (16), and two
molecular mass forms of this peptidase have been described (17, 18).
This enzyme(s) has also been shown to possess the ability to hydrolyze
partially degraded type I collagen, releasing the Gly-Pro dipeptide,
and it was suggested that, in collaboration with collagenase,
dipeptidyl peptidase IV may contribute to the destruction of the
periodontal ligament (19). In addition to this potential pathological
function, glycylprolyl peptidase may also play a vital role in
providing P. gingivalis with dipeptides, which can be
transported inside the cell and serve as a source of carbon, nitrogen,
and energy for this asaccharolytic organism. Recently, a gene encoding
glycylprolyl peptidase has been cloned and sequenced, and it is now
apparent that this enzyme is homologous to dipeptidyl peptidase IV from
other organisms (20). In this report we describe the purification and
characterization of another cell surface-associated serine proteinase
of P. gingivalis with a novel prolyl tripeptidyl peptidase
activity but with a surprising primary structure related to dipeptidyl
peptidases. This enzyme liberates tripeptides from the free amino
terminus and possesses the absolute requirement for the proline residue in the P1 position.
Materials
Diisopropylfluorophosphate
(DFP),3 leupeptin, and
3,4-dichloroisocoumarin were purchased from Calbiochem. Antipain,
iodoacetamide, substance P, bradykinin, and bradykinin-related peptides
were obtained from Sigma. Other peptides used in this study were
synthesized at the Molecular Genetic Instrumental Facility using an
Fmoc (N-(9-fluorenyl)methoxycarbonyl) protocol with an
advanced ChemTech MPS350 automated synthesizer. H-Ala-Phe-Pro-pNA,
H-Gly-Pro-pNA, Z-Gly-Pro-pNA, Z-Ala-Pro-pNA, and H-Pro-pNA were
obtained from Bachem. Prolinal was kindly provided by Dr. James Powers
(Georgia Institute of Technology, Atlanta, GA), and cystatin C was
provided by Dr. Magnus Abrahamson (University of Lund, Lund, Sweden).
Methods
Source and Cultivation of Bacteria--
P. gingivalis
HG66 was a gift of Dr. Roland Arnold (University of North Carolina,
Chapel Hill, NC), and the strains W50 and ATCC 33277 were obtained from
the American Type Culture Collection. All cells were grown as described
previously (21).
Enzyme Activity Assays--
Routinely, the tripeptidyl peptidase
amidolytic activity was measured with H-Ala-Phe-Pro-pNA (1 mM) in 0.2 M HEPES, pH 7.5, at 37 °C. The
assay was performed in a total volume of 0.2 ml on microplates, and the
initial turnover rate was recorded at 405 nm using a microplate reader
(Spectramax, Molecular Devices). In inhibition studies, the enzyme was
first preincubated with inhibitor for 15 min at 37 °C, substrate was
added, and residual activity was recorded. H-Gly-Pro-pNA,
Z-Ala-Pro-pNA, Z-Gly-Pro-pNA, and H-Pro-pNA (1 mM final
concentration) were assayed in the same manner.
Protein Determination--
Protein concentration was determined
using the BCA reagent kit (Sigma), using bovine serum albumin as a standard.
Localization of Tripeptidyl Peptidase Activity--
Cultures of
P. gingivalis HG66, W50, and ATCC 33277, at different phases
of growth, were subjected to the following fractionation procedure. The
cells were removed by centrifugation (10,000 × g, 30 min), washed twice with 10 mM Tris, 150 mM
NaCl, pH 7.4, resuspended in 50 mM Tris, pH 7.6, and
disintegrated by ultrasonication in an ice bath at 1500 Hz for 5 cycles
(5 min of sonication and 5 min of brake). Unbroken cells and large
debris were removed by centrifugation (10,000 × g, 30 min), and the opalescent supernatant was subjected to
ultracentrifugation (150,000 × g, 120 min), yielding a
pellet containing bacterial membranes and a supernatant that was
considered membrane-free cell extract. All fractions, as well as the
full culture, culture medium, and full culture after sonication, were
assayed for amidolytic activity against H-Ala-Phe-Pro-pNA.
Enzyme Purification--
All purification steps were performed
at 4 °C except for FPLC separations, which were carried out at room
temperature. Cells were harvested by centrifugation (6000 × g, 30 min), washed with 50 mM potassium
phosphate buffer, pH 7.4, and resuspended in the same buffer (150 ml/50
g of cells, wet weight). Triton X-100 (10% v/v in H2O) was
added slowly to the bacterial cell suspension to a final concentration
of 0.05%. After 120 min of gentle stirring, unbroken cells were
removed by centrifugation (28,000 × g, 60 min).
Proteins in the supernatant were precipitated with cold acetone
( Electrophoretic Techniques--
The SDS-PAGE system of Schagger
and von Jagow (22) was used to monitor enzyme purification and estimate
the enzyme molecular mass. For amino-terminal sequence analysis,
proteins resolved in SDS-PAGE were electroblotted to polyvinylidene
difluoride membranes using 10 mM
3-(cyclohexylamino)propanesulfonic acid, pH 11, 10% methanol (23). The
membrane was washed thoroughly with water and stained with Coomassie
Blue G250. The blot was air dried, and protein bands were cut out and
subjected to amino-terminal sequence analysis with an Applied
Biosystems 491 protein sequencer using the program designed by the manufacturers.
Enzyme Fragmentation--
The purified prolyl tripeptidyl
peptidase (PTP-A) was partially denatured by incubation in 6 M urea in 0.02 M Tris, pH 7.6, for 60 min. Low
molecular mass gingipain R (RgpB) from P. gingivalis was
then added to make an enzyme:substrate molar ratio of 1:100; the
reaction mixture was made in 1 mM cysteine; and the sample was incubated overnight at 37 °C. Generated peptides were separated by reverse phase HPLC using a µBondapak C-18 column (3.9 × 300 mm; Waters, Milford, MA). Peptides were eluted with 0.1%
trifluoroacetic acid and acetonitrile containing 0.08% trifluoroacetic
acid, using a gradient from 0 to 80% acetonitrile over 60 min.
Peptides were monitored at 220 nm and collected manually.
For determination of the active site serine residue and to confirm that
the purified enzyme was, indeed, a serine proteinase, 100 µg of
purified PTP-A was first incubated with 170 µCi of
[1,3-3H]DFP (Amersham Pharmacia Biotech) for 30 min at
25 °C in 20 mM HEPES, pH 7.5. The reaction was quenched
by addition of cold DFP to a final concentration of 10 mM,
and the radiolabeled material was analyzed by SDS-PAGE, followed by
autoradiographic analysis. The gel was dehydrated, soaked in
2,5-diphenyloxazole solution for 2 h, and dried, and the
DFP-binding proteins were detected by fluorography after an exposure
time of 96 h on x-ray film (XAR; Eastman Kodak). The bulk of
radiolabeled protein was subjected to proteolytic fragmentation with
RgpB, and peptides obtained were separated by reverse phase HPLC as
described above. Radioactivity in each peptide fraction was measured
using a Identification of the PTP-A Gene--
The data base containing
the unfinished P. gingivalis W83 genome, available from The
Institute for Genomic Research, was searched for the presence of
nucleotide sequences corresponding to the amino-terminal and the
internal PTP-A amino acid sequences using the TBALSTN algorithm (24).
An identified clone, gnl|TIGR|P. gingivalis_126, was
retrieved from The Institute for Genomic Research (TIGR) data base. The
position of the PTP-A gene was localized using the National Center for
Biotechnology Information open reading frame (ORF) finder, and the
amino acid sequence, obtained by conceptual translation of the entire
ORF, was further used for homology screening by use of the NCBI BLAST
search tool.
Enzyme Specificity--
Peptides were incubated with 1 µg of
PTP-A at an enzyme:substrate molar ratio of 1:100 for 3 or 24 h in
50 µl of 200 mM HEPES, pH 7.5, at 37 °C, and the
reaction was stopped by acidification with trifluoroacetic acid. The
samples were then subjected to reverse phase high pressure liquid
chromatography using a µBondapak C-18 column (3.9 × 300 mm)
(Waters, Milford, MA) and an acetonitrile gradient (0-80% in 0.075%
trifluoroacetic acid in 50 min). Each peak, detected at 220 nm, was
collected, lyophilized, redissolved in 50% (v/v) methanol, 0.1%
acetic acid, and subjected to analysis by mass spectrometry.
Mass Spectrometry--
A Finnigan MAT 95S sector mass
spectrometer (Finnigan MAT, Bremen, Germany) equipped with an
electrospray source (ESI) was operated essentially as described
previously (25). Peptides were identified by fitting of the obtained
spectra to specific sequences using an Internet application program
MsFit available at http://falcon.ludwig.ucl.ac.uk/msfit.html.
Enzyme Localization, Purification, and Initial
Characterization--
Analysis of amidolytic activity against
H-Ala-Phe-Pro-pNA in several fractions of P. gingivalis,
HG66, W50, and ATCC 33277, clearly indicated that an enzyme(s) with
prolyl tripeptidyl peptidase activity is localized on the cell surface
in all strains tested, with <5% of the total activity being found in
the medium regardless of the growth phase of the bacterial culture.
Cell-associated enzyme was easily detached from the bacterial surface
by treatment with a low concentration (0.05%) of Triton X-100. This
procedure released >85-90% of activity in a soluble form. Subsequent
acetone precipitation of proteins in the Triton X-100 fraction
successfully separated the activity from pigment that remained in
solution. The redissolved protein fraction, after dialysis, was applied to hydroxyapatite chromatography, and at this step substantial separation of the PTP-A activity from both the dipeptidyl peptidase IV
and bulk protein was achieved (Fig.
1A). Further purification performed by subsequent chromatography steps, including
phenyl-Sepharose (Fig. 1B), MonoQ (Fig. 1C), and
MonoP columns (Fig. 1D), resulted in the isolation of
purified enzyme. Significantly, the chromatography step on MonoP
yielded an A280 profile much sharper than the
activity peak. Although this imperfect overlap of protein and activity profiles may suggest that the protein component does not represent active enzyme, the rest of data argue with such a contention. This
apparent contradiction may be likely explained by the enzyme inhibition
by the reaction product of H-Ala-Phe-Pro-pNA hydrolysis, but this
possibility has not been been explored. The yield of protein and
activity recovery in a typical purification procedure is summarized in
Table I.
SDS-PAGE analysis of the purified enzyme revealed the presence of two
protein bands with apparent molecular masses of 81.8 and 75.8 kDa,
respectively (Fig. 2, lane f).
Autoradiography of the enzyme sample radiolabeled with
[1,3-3H]DFP (Fig. 2, lane g) clearly indicated
that the bands represented either two distinct serine proteinases or
different molecular mass forms of the same enzyme. In an attempt to
distinguish between these two options, the electrophoretically resolved
proteins were subjected to amino-terminal sequence analysis, but,
unfortunately, it was found that the 81.8-kDa form of PTP-A had a
blocked amino terminus. In contrast, the sequence
NH2-SAQTTRFSAADLNALMP- was found at the amino terminus of
the lower molecular mass form of the enzyme. This result led us to the
possibility that the 75.8-kDa form of PTP-A was derived from the
81.8-kDa form through proteolytic cleavage of a 6-kDa amino-terminal
peptide. To confirm this hypothesis and, in addition, to localize the
active site residue within P. gingivalis PTP-A, the mixture
containing both radiolabeled enzymes was proteolytically fragmented,
and peptides were resolved by reverse phase HPLC. This procedure
yielded only one major radioactive peptide peak (data not shown), and
the purified peptide was found to have a single amino acid sequence:
IGVHGWXYGGFMTTNL, where X apparently represents
the DFP-modified serine residue. These data convincingly indicate that
the two protein bands of purified PTP-A represents different forms of
the same enzyme.
pH Optimum, Stability, and Inhibition Profile--
Using the
amidolytic activity assay with H-Ala-Phe-Pro-pNA, it was found that the
enzyme has a broad pH optimum from pH 6.0 to 8.0, and in 0.2 M HEPES, pH 7.6 was stable for at least 12 h at 25 or
37 °C.
PTP-A activity was not affected by class-specific synthetic inhibitors
of cysteine or metalloproteinases (Table
II). In contrast, preincubation of the
enzyme with DFP or Pefablock resulted in total loss of activity,
supporting its classification as a serine proteinase. Surprisingly,
however, 3,4-dichloroisocumarin was only a poor inhibitor, and
phenylmethylsulfonyl fluoride, leupeptin, antipain, and prolinal had no
effect at all. Interestingly, preincubation of PTP-A with
iodoacetamide, but not with N-ethylmaleimide, stimulated enzyme amidolytic activity ~2-fold. Human plasma inhibitors, such as
Substrate Specificity--
Among several chromogenic substrates
tested, including H-Ala-Phe-Pro-pNA, H-Gly-Pro-pNA, Z-Gly-Pro-pNA,
Z-Ala-Pro-pNA, and H-Pro-pNA, only H-Ala-Phe-Pro-pNA was hydrolyzed by
PTP-A, indicating a prolyl-specific tripeptidyl peptidase activity. To
further confirm this specificity, several synthetic peptides composed
of 5-34 amino acid residues and containing at least 1 proline residue were tested as substrates for PTP-A. Of 22 peptides tested only those
with a proline residue in the third position from the amino-terminal end were cleaved (Table III), with the
significant exception of peptides with adjacent proline residues
(peptides 3, 4, and 16). In addition, a free
The lack of cleavage after internal proline residues in the synthetic
peptides corresponds well with the absence of any proteolytic activity
on several protein substrates, including IgA, IgG, albumin, azocasein,
carboxymethylated ribonuclease, and gelatin. However, the size of the
substrate, which is a limiting factor in the activity of
oligopeptidases (26), is not restricting in the case of PTP-A, because the enzyme is able to cleave a tripeptide
(NH2-Xaa-Xaa-Pro) from the amino terminus of both human
cystatin C and interleukin 6 (data not shown).
PTP-A Sequence Analysis--
Partial PTP-A amino acid sequence
data allowed us to identify the P. gingivalis genomic clone
gnl|TIGR|P. gingivalis_126 in the Unfinished Microbial
Genomes data base, TIGR. An ORF corresponding to the PTP-A amino acid
sequence was found, as indicated by the fact that all sequences of the
PTP-A-derived peptides obtained by the enzyme polypeptide fragmentation
with RgpB were present in the protein primary structure inferred from
the nucleotide sequence of the ORF. The 732-amino acid polypeptide with
a calculated mass of 82,266 Da was encoded in this ORF. The homology
search performed using the NCBI TBLASTN tool against GenBank, EMBL,
DDBJ, and PDB data bases, and subsequent multiple sequence alignments (Fig. 3) indicated that PTP-A is a new
member of the prolyl oligopeptidase family of serine proteinases (27).
Within this large and diverse family of evolutionary and functionally
related enzymes both from prokaryotes and eukaryotes, PTP-A was most
closely related to bacterial dipeptidyl peptidase IV from
Flavobacterium meningosepticum, Xantomonas
maltophilus, and P. gingivalis, sharing 31.6, 30.4, and
28.5% amino acid sequence identity, respectively. Remarkably, the
carboxyl-terminal region of the PTP-A molecule (residues 502-732) shows a significant similarity to the eukaryotic prolyl
oligopeptidases, with 34 and 33% identity to human dipeptidyl
peptidase IV and mouse fibroblast activation protein (FAP),
respectively (Fig. 3). This part of the molecule contains the amino
acid residues that encompass the catalytic triad in all characterized
prolyl oligopeptidases, and from the multiple alignments with
dipeptidyl peptidase IV of confirmed active site residues (28) it is
apparent that Ser-603, Asp-768, and His-710 represent the catalytic
triad of PTP-A (Fig. 3). Such an inference is further supported by the direct labeling of Ser-603 by DFP. In addition, the computer-assisted search for sequential motifs characteristic for transmembrane domains
revealed the presence of such a putative region within the
amino-terminal sequences of PTP-A, with residues 5-25 most likely
folded into a hydrophobic Because of their cyclic aliphatic character, proline residues
bestow unique conformational constraints on polypeptide chain structure, significantly affecting the susceptibility of proximal peptide bonds to proteolytic cleavage (29). It is assumed, therefore, that those proline residues that often appear near the amino termini of
many biologically active peptides protect them against proteolytic degradation by proteinases with general specificity. For the cleavage of peptide bonds adjacent to proline residues a specialized group of
proteolytic enzymes has evolved, and their activity in vivo may have important physiological significance, because it may lead to
inactivation of many biologically active peptides and/or transformation
of the activity of others (30). In addition, hydrolysis of prolyl-X
bonds in conjunction with general catabolic pathways should allow the
complete reutilization of amino acids by living organisms, including
bacteria. However, prolyl peptidases from bacterial pathogens, if
released into the host environment, may interfere with the
physiological functions of biologically active peptides and proteins
and, therefore, contribute to the pathogenecity of infectious disease.
In this report we have described the purification, properties, and
amino acid sequence of a novel prolyl-specific tripeptidyl peptidase
from P. gingivalis. PTP-A releases tripeptides from small
proteins such as interleukin 6 and cystatin C as well as from peptides.
It works as an exopeptidase because those peptides with a blocked
PTP-A was classified as a serine protease based on its inhibition by
DFP and its resistance to sulfydryl blocking and chelating agents, and
this was also confirmed by sequencing of the peptide containing the
DFP-labeled active site residue. The sequence GWSYGG is the signature
for a recently identified group of serine proteinases, the prolyl
oligopeptidase (S9) family, and indeed, sequence alignment clearly
indicates that PTP-A is a new member of this family, most closely
related to bacterial and eukaryotic dipeptidyl peptidases IV. In
parallel with other members of the S9 family, P. gingivalis PTP-A contains the typical prolyl oligopeptidase catalytic triad topology of Ser-603-Asp-678-His-711 located at the carboxyl-terminal end of the amino acid sequence in a region now designated the protease domain.
Despite structural similarities to proteinases from the prolyl
oligopeptidase family, the tripeptidyl peptidase activity of PTP-A is
unusual for this group of enzymes, and no other known similar activity
has so far been attributed to any other member of the S9 family. In
fact, all strict tripeptidyl peptidases belong only to the subtilisin
family (S8) and the S33 family of serine proteinases; however, they
neither share a structural relationship with PTP-A nor have activity
limited to cleavage after proline residues (31). In this respect, the
P. gingivalis tripeptidyl peptidase is a unique enzyme, and
it will be interesting to learn the structural basis for its narrow specificity.
In P. gingivalis PTP-A, as well as in dipeptidyl peptidase
IV (19), all activities are cell surface associated, and it is conceivable that the enzymes are membrane anchored through putative signal sequences, which are not cleaved but remain as a
membrane-spanning domain similar to other members of the prolyl
oligopeptidase family (32). However, a significant portion of the
purified PTP-A has a truncated amino terminus, apparently attributable
to cleavage by Lys-specific proteinase and likely to be an artifact
that has occurred during the purification procedure. Nevertheless, the proteolytic shedding of membrane-bound PTP-A and dipeptidyl peptidase IV also occurs during cultivation of the bacteria, as indicated by
variable amount of soluble activities found in cell-free culture media.
The cell surface localization of dipeptidyl and tripeptidyl peptidases
also supports a putative physiological function in providing nutrients
for growing bacterial cells. Here, the inability of asaccharolytic
P. gingivalis to utilize free amino acids (34) makes the
bacterium entirely dependent on an external peptide supply. In this
regard, dipeptidyl peptidase IV and PTP-A activities are probably very
important, if not indispensable, for bacterial growth. This suggestion
is strongly corroborated by the fact that the P. gingivalis
genome contains three additional genes encoding proteinases homologous
with dipeptidyl peptidase IV and PTP-A and one related to
aminopeptidase B. If expressed, each gene product would probably have
enzymatic activity, because each has a well preserved catalytic triad
(Fig. 4). In addition, all of these genes
encode a putative signal peptide, which may act in providing membrane
anchorage motifs. Whether each of these enzymes is constitutively expressed or forms a backup system to provide nutrients in a specific environment is presently unknown, but our preliminary data
indicate that at least one aminopeptidase
B homologue, also having tripeptidyl peptidase activity, is produced
during P. gingivalis growth.4
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C) added to a final concentration of 60%, collected by
centrifugation, redissolved in 50 mM potassium phosphate
buffer, pH 7.0, and extensively dialyzed against 20 mM
potassium phosphate, pH 7.0, containing 0.02% sodium azide. The
dialyzed fraction was clarified by centrifugation (28,000 × g, 30 min) and applied to a hydroxyapatite column (Bio-Rad)
equilibrated with 20 mM potassium phosphate, pH 7.0, at a
flow rate of 20 ml/h, after which the column was washed until the
A280 fell to zero. Bound proteins were eluted
with a gradient from 20 to 300 mM potassium phosphate, and
fractions (7 ml) were analyzed for dipeptidyl- and tripeptidyl peptidase activity using H-Gly-Pro-pNA and H-Ala-Phe-Pro-pNA, respectively. The activity against the latter substrate was pooled, saturated with 1 M ammonium sulfate, clarified by
centrifugation, and directly loaded onto a phenyl-Sepharose HP
(Amersham Pharmacia Biotech) column equilibrated with 50 mM
potassium phosphate, pH 7.0, containing 1 M ammonium
sulfate. The column was washed with two volumes of equilibration
buffer, followed by buffer containing 0.5 M ammonium
sulfate, and developed with a descending gradient of ammonium sulfate
from 0.5 to 0 M. Active fractions were pooled, extensively
dialyzed against 20 mM Tris, pH 7.5, and applied to a MonoQ
HR 5/5 FPLC column equilibrated with the same buffer. The column was
washed with 5 volumes of equilibration buffer at 1.0 ml/min, after
which bound proteins were eluted with a gradient of 0-300
mM NaCl. The active fractions were pooled, dialyzed against 25 mM Bis-Tris, pH 6.3, and subjected to chromatofocusing
on a MonoP FPLC column equilibrated with Bis-Tris buffer, using a pH gradient developed with 50 ml of 10 × diluted Polybuffer 74 (Amersham Pharmacia Biotech) adjusted to a pH of 4.0.
liquid scintillation counter, and the labeled peptide as
well as other selected peptides were subjected to sequence analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of the prolyl tripeptidyl
peptidase from the acetone precipitate of the P. gingivalis
cell extracts. , absorbance at 280 nm;
, amidolytic activity
against AFP-pNA;
, amidolytic activity against GP-pNA. A,
separation of PTP-A on hydroxyapatite (100 ml) equilibrated with 20 mM potassium phosphate buffer, pH 7.0. The elution was
carried out with 20 mM potassium phosphate buffer, pH 7.0, using a phosphate gradient from 20 to 300 mM at flow rate
20 ml/h. B, separation of PTP-A obtained from previous step
on phenyl-Sepharose HP (25 ml) equilibrated with 50 mM
potassium phosphate, 1 M ammonium sulfate, pH 7.0, at flow
rate of 30 ml/h. The column was washed with 2 volumes of equilibration
buffer, and a step gradient of 0.5 M ammonium sulfate was
applied, after which a descending gradient of 0.5-0 M
ammonium sulfate was applied. C, separation of PTP-A on a
MonoQ FPLC column. The PTP-A-containing fractions were extensively
dialyzed against 20 mM Tris-HCl, pH 7.0, concentrated by
ultrafiltration, and applied to a MonoQ column equilibrated with the
same buffer. The column was washed with 5 volumes of equilibration
buffer, after which bound protein was eluted with a gradient of 0-300
mM NaCl. D, chromatofocusing of PTP-A on MonoP.
The concentrated fraction of PTP-A from the previous step, equilibrated
with 25 mM Bis-Tris, pH 6.3, was loaded on a MonoP column
equilibrated with the same buffer. A pH gradient was developed using 50 ml of Polybuffer 74, with the pH adjusted to 4.0.
Purification of the PTP-A from P. gingivalis
View larger version (92K):
[in a new window]
Fig. 2.
SDS-PAGE of fractions from purification of
PTP-A and the autoradiography of the purified enzyme. Lane
a, molecular mass markers (phosphorylase B, 97 kDa; bovine serum
albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean
trypsin inhibitor, 20 kDa; -lactalbumin, 14 kDa); lane b,
acetone precipitate from Triton X-100 extract of P. gingivalis; lane c: hydroxyapatite column eluate;
lane d, phenyl-Sepharose column eluate; lane e,
MonoQ column eluate; lane f, purified PTP-A from MonoP
column wash; lane g, autoradiograph of
[3H]DFP-labeled enzyme exposed for 96 h to x-ray
film. All samples were reduced and boiled before PAGE analysis.
1-proteinase inhibitor,
1-antichymotrypsin, and
2-macroglobulin, did not affect the enzyme activity, nor were they cleaved by PTP-A (data not shown).
Effect of inhibitors on the amidolytic activity of PTP-A
-amino group was
absolutely required for cleavage after the third proline residue, as
exemplified by resistance to enzymatic hydrolysis of peptide 9, which
differs from peptide 8 only in acylation of the
-amino group of the
amino-terminal valine residue. Except for these two limitations, the
peptide bond -Pro-
-Yaa- was cleaved at the same rate in all peptides with the general formula
NH2-Xaa-Xaa-Pro-Yaa-(Xaa)n, where Xaa represents any amino acid residue, whereas Yaa could be any residue except proline, regardless of the chemical nature of the amino acids
and the length of the peptide. In all cases the reaction was completed
within 3 h, and prolonged incubation for 24 h did not affect
the pattern of cleavage, confirming the absolute requirement for a
proline residue at the third position from the unblocked amino
terminus. In addition, these data indicate that the preparation of
PTP-A was free of any contamination with aminopeptidase, dipeptidyl peptidase, or endopeptidase activities.
Cleavage specificity of tripeptidyl peptidase on synthetic peptides
-helix responsible for membrane anchoring
of this enzyme.
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Fig. 3.
Multiple sequence alignment of P. gingivalis PTP-A (Pg-PTP-A) and its bacterial and
eukaryotic homologues. Sequences of dipeptidyl peptidase
from P. gingivalis (Pg-DPP; Ref. 20; note
that the Pg-DPP amino-terminal sequence was corrected
according to the data from the P. gingivalis W83 genome),
dipeptidyl peptidase from Flavobacterium meningosepticum
(Fm-DPP), human dipeptidyl peptidase IV
(Hs-DPP), and mouse fibroblast activation protein
(Mm-FAP) were aligned using the ClustalW multiple sequence
alignment tool. Peptide sequences obtained from PTP-A analysis are
indicated with arrows (note that the sequence of the peptide
81-97 corresponds to the amino terminus of the lower molecular weight
form of PTP-A); catalytic triad is marked with asterisks;
and the proposed PTP-A membrane-anchoring amino-terminal -helix is
underlined. Homologous regions are
highlighted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group are not processed. In addition, the enzyme has an
absolute requirement for Pro in the P1 position because the
substitution of proline by any other amino acid, including hydroxyproline, is not tolerated.
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Fig. 4.
Comparison of P. gingivalis PTP-A
and dipeptidyl peptidase active site domains to corresponding sequences
of three putative homologues identified within the P. gingivalis genome (DPP-H1, DPP-H2, and
DPP-H3). Sequences of P. gingivalis
PTP-A, DPP, DPP-H1, DPP-H2, and DPP-H3 were obtained from
conceptual translation of the following ORFs retrieved from TIGR
unfinished P. gingivalis genome data base:
gnl|TIGR|P. gingivalis_126 (positions 13,228-15,426),
_87 (positions 6424-4399), _65 (positions 161-1786), _101 (positions
8895-6845), and _9 (positions 4216-2162), respectively. The sequences
were subsequently aligned using the ClustalW multiple sequence
alignment tool. Residues predicted as catalytic triads of serine
proteinases are marked with asterisks.
Although there is a little doubt that P. gingivalis
homologues of prolyl peptidases can be considered important
housekeeping enzymes, their external localization and uncontrolled
activity may contribute significantly to runaway inflammation in the
human host and the pathological degradation of connective tissue during periodontitis. Working in concert, the bacterial PTP-A and dipeptidyl peptidase IV have the ability to completely degrade collagen fragments locally generated be endogenous or bacterial collagenases. Because type
I collagen is the major component of periodontal ligament, its enhanced
degradation by dipeptidyl peptidase IV and PTP-A, as well as by
functionally related enzymes released by other periodontopathogens (33), may contribute to tooth attachment loss and periodontal pocket
formation; thus these enzymes must be considered important pathogenic
factors of these bacteria.
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ACKNOWLEDGEMENTS |
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Sequence data for PTP-A was obtained from The Institute for Genomic Research website at http://www.tigr.org. We thank Dorota Panek for superb technical assistance and Elzbieta Stankiewicz and Tomasz Dec for help.
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FOOTNOTES |
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*
This work was supported by Grant 6PO4C 083 18 from the
Committee of Scientific Research (Komitet Bada Naukowych,
Poland; to J. P. and A. B.) and National Institutes of Health
Grant DE 09761 (to J. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602. Tel.: 706-542-1713; Fax: 706-542-3719; E-mail:
potempa{at}arches.uga.edu.
1 T. Imamura, A. Banbula, P. J. B. Pereira, J. Travis, and J. Potempa, submitted for publication.
2 D. Nelson, J. Potempa, T. Kordula, and J. Travis, submitted for publication.
4 A. Banbula, M. Bugno, J. Travis, and J. Potempa, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DFP, diisopropylfluorophosphate; PTP-A, prolyl tripeptidyl peptidase A; pNA, p-nitroanilide; Z, benzyloxycarbonyl; H, hydroxyproline; FPLC, fast performance liquid chromatography; PAGE, polyacrylamde gel electrophoresis; HPLC, high pressure liquid chromatography; ORF, open reading frame; TIGR, The Institute for Genomic Research; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
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REFERENCES |
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