From the Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Georgia 30602 and the Departments of
Microbiology and Immunology and § Animal
Biochemistry, Jagiellonian University, 31-120 Krakow, Poland
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ABSTRACT |
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Periodontal disease is characterized by
inflammation of the periodontium manifested by recruitment of
neutrophils, which can degranulate, releasing powerful proteinases
responsible for destruction of connective tissues, and eventual loss of
tooth attachment. Although the presence of host proteinase inhibitors
(serpins) should minimize tissue damage by endogenous proteinases, this is not seen clinically, and it has been speculated that proteolytic inactivation of serpins may contribute to progression of the disease. A
major pathogen associated with periodontal disease is the Gram-negative anaerobe Porphyromonas gingivalis, and in this report, we
describe a novel proteinase that has been isolated from culture
supernatants of this organism that is capable of inactivating the human
serpin, The anaerobe Porphyromonas gingivalis has been strongly
implicated as a major causative organism of adult onset periodontal disease (1-4). Enzymes from this organism have been found to degrade
collagen, fibrinogen, immunoglobulins, complement proteins, and
fibronectin, among others (for reviews, see Refs. 5-8). Recent evidence has shown that three proteinases released from P. gingivalis, referred to as gingipains R (Rgp A and Rgp
B)1 and gingipain K because
of their ability to cleave specifically after arginine and lysine
residues, respectively, may have a more physiologically relevant role
in modulating the human immune system rather than in their general
ability to degrade proteins. Working in concert, these proteinases have
already been shown to produce bradykinin from high molecular weight
kininogen, either directly or indirectly (kallikrein activation),
resulting in vascular permeability enhancement (9). This mechanism,
which is used to provide nutritional components for the growth and
proliferation of P. gingivalis, is presumed to be
responsible for both the increased gingival crevicular fluid (GCF) and
edema noted clinically in the periodontal pockets of patients with
advanced periodontitis (10).
The interaction of P. gingivalis with the host innate immune
response have been paradoxical, with both pro- and anti-inflammatory responses reported. For example, P. gingivalis
lipopolysaccharide has been shown to increase mRNA levels of
interleukin 8 in neutrophils (11), whereas gingipains R have been shown
to increase neutrophil chemotaxis by release of C5a from C5 of the
complement system (12). However, these same proteinases are also
capable of cleaving the C5a receptor from infiltrating neutrophils
(13), effectively neutralizing their localized chemotactic activity.
Additionally, P. gingivalis cells have the ability to
inhibit both interleukin 8 accumulation in gingival epithelial cells
(14) as well as transepithelial migration (15). These contradictions
may be explained by an apparent compartmentalization in the periodontal cavity, whereby distal activation of chemotactic components and proximal paralysis of these same factors create a "leukocyte wall" between the periodontal plaque and gingival epithelium (16). Indeed, it
has recently been reported that soluble gingipains can stimulate
interleukin 8 activity, whereas membrane-bound gingipains, with a
limited ability to diffuse beyond the plaque surface, completely degrade interleukin 8 (17).
The recruitment of neutrophils to the leukocyte wall through both the
increased leakage of blood vessels and a chemotactic gradient would at
first seem suicidal to P. gingivalis. However, this is not
likely to be the case, as this organism has evolved mechanisms to
survive in the presence of neutrophils. P. gingivalis proteinases have been shown to degrade C3 of the complement system and
immunoglobulins (18, 19), thereby averting opsonization and subsequent
detection by the host. Furthermore, gingipain R has been shown to have
an inhibitory effect on the oxidative burst utilized by neutrophils to
kill microorganisms (20). Similarly, the bacterial outer membrane of
P. gingivalis can act as an antioxidant sink due to the
incorporation of large amounts of heme (21).
Activated neutrophils in the leukocyte wall undergo degranulation, due
to the inability to phagocytize foreign organisms, thereby expelling
large quantities of human neutrophil elastase (HNE) and cathepsin G. Although these proteinases can cause abnormal connective tissue
destruction, the presence of human plasma proteinase inhibitors
(serpins) should minimize this process as they would complex with the
endogenous proteinases and be taken up by the liver for degradation. In
fact, high protein levels of Recently, we provided preliminary data that indicated that P. gingivalis elicited a proteinase that rapidly inactivated
Materials Used
Diisopropyl fluorophosphate, leupeptin, and
3,4-dichloroisocoumarin were purchased from Calbiochem. All other
materials used were obtained from Sigma, unless otherwise indicated,
and were of at least analytical grade.
Methods
Bacteria Cultivation--
The strain of P. gingivalis
(HG66) that was used for the purification of periodontain was a gift of
Dr. Roland Arnold (University of North Carolina, Chapel Hill, NC).
Cells were grown in 5 liters of broth containing 150 g of
trypticase soy broth, 25 g of yeast extract (both from Difco), 25 mg of hemin, 2.5 g of cysteine, 0.5 g of dithiothreitol, and
5 mg of menadione (all from Sigma), anaerobically, at 37 °C for
24 h in an atmosphere of 85% N2, 10% CO2, 5% H2. The seed culture was used to
inoculate 100 liters of the same broth, and the bacterium was then
grown in a 130-liter fermentor (W.B. Moore, Inc.) at the University of
Georgia Fermentation Plant. Cells were grown for 24 h until late
stationary phase of bacterial growth (A660 > 2.0). Additional strains, 33277, W50 (both from ATCC), W12, and 381 (both gifts of Dr. Caroline A. Genco, Boston University Medical
School), were grown under the same conditions in 1-liter volumes.
Cellular Localization--
Whole cell culture mixtures were
fractionated to determine localization of periodontain. First, a low
speed centrifugation (6,000 × g for 20 min at 4 °C)
was used to pellet the cells, after which the supernatant was subjected
to high speed ultracentrifugation (100,000 × g for 120 min at 4 °C) to separate vesicles from the supernatant, which
contained all soluble proteins. For quantitation of proteolytic
activity all samples were brought to an equal volume by the addition of
50 mM Tris, pH 7.4.
Proteinase Purification--
Bacteria were harvested at
stationary phase, and the cells were removed by continuous
centrifugation (Sharples AS-16P). The cell-free culture fluid was
concentrated to 5 liters in a Pellicon system (Millipore), using a
30-kDa molecular mass cut-off membrane, and then precipitated with 7.5 liters of acetone at Electrophoresis--
Enzyme purification and visualization of
the heavy and light chains was monitored by SDS-PAGE on a 10%
separating gel using the Tris-HCl/Tricine buffer system, according to
Schagger and von Jagow (28). Nondenaturing PAGE (29) in a 4-20%
gradient gel was used to show the native protein as a single band.
Molecular Mass Determination--
The mass of the native enzyme
was determined by gel filtration using a TSK-GEL G3000SW (TosoHaas)
calibrated with gel filtration standards (Bio-Rad). The mass of the
separated heavy and light chains from SDS-PAGE were estimated by
scanning the gel using the Eagle Eye II imaging system (Stratagene) and
calculating a linear regression of low molecular weight electrophoresis
standards (Amersham Pharmacia Biotech) as reference. Accurate molecular mass measurements of the digestion products of Protein Sequence Analysis--
For amino-terminal sequence
analysis, proteins resolved by electrophoresis were electrotransferred
onto a polyvinylidene difluoride membrane according to Matsudaira (30).
Sequence analysis was performed with an Applied Biosystems 4760A
gas-phase sequencer at the Molecular Genetics Instrumentation Facility
(University of Georgia, Athens, Ga) operated according to the
manufacturer's recommendations.
Inhibition Studies--
For inhibition studies, periodontain was
used at a concentration that was capable of completely inactivating
0.15 nmol of Protein and Peptide Degradation by Periodontain--
The
degradation of proteins was followed by using either native or reduced,
carboxymethylated, maleylated lysozyme (30 µM). Either protein was incubated with periodontain (30 nM) in a
final volume of 20 µl of assay buffer for specific time intervals.
The reaction was stopped by addition of 20 µl of SDS sample buffer (4% SDS, 20% glycerol, 0.125 M Tris-HCl, pH 6.8),
followed by boiling for 5 min, after which the entire sample was
electrophoresed on a 12% gel and stained in 0.1% Coomassie Brilliant
Blue to visualize the protein bands.
To determine the cleavage sites within the reactive site loop (RSL) of
For analysis of the fragments obtained through digestion of the insulin
Gelatin Zymograph--
Zymography analysis on gelatin gels was
performed on pure samples of periodontain in the presence of 5 mM cysteine, with or without 100 µM E-64.
After the addition of SDS sample buffer, the samples were subjected to
electrophoreses at 4 °C on a 10% SDS-PAGE with gelatin (Difco) (0.1 mg/ml) incorporated into the gel. Following electrophoresis, the gel
was washed twice with 2.5% (w/v) Triton X-100 to remove the SDS and
then incubated in activation buffer (50 mM Tris, 20 mM cysteine, pH 7.4) at 37 °C for 2 h. The
zymograph was developed in 0.1% Amido Black, with clearing zones
indicating proteolytic digestion of the incorporated gelatin.
Cloning of Gene Fragment Encoding the Amino Terminus of
Periodontain--
Based on the amino-terminal sequence (23 residues)
of the heavy chain (55 kDa) of periodontain, a pair of degenerate
oligonucleotide primers (5'-ACNGA(G/A)GGNGTNCCNGC-3') and
(5'-NCGCATNGG(G/A)TC(G/A)TT-3') corresponding to residues 1-6 (TEGPA)
and 19-23 (NDPMR), respectively, were designed. The DNA fragment
coding for the amino terminus of periodontain was amplified by PCR
using Pwo DNA polymerase (Roche Molecular Biochemicals) and
10 ng of W50 P. gingivalis DNA (purified by the Purgene kit,
Gentra Systems Inc.). PCR was carried out with 500 ng of primers for 1 min at 94 °C, 1 min at 65 °C, and 20 s at 72 °C. The
expected product of 69 base pairs was purified from a 2% agarose gel
with the Ultrafree MC Millipore filter (Millipore), phosphorylated at
the 5'-end with polynucleotide kinase, and blunt end ligated into a
SmaI-digested pUC19 vector. The fragment coding for the
amino terminus of periodontain was identified by sequence analysis.
Identification of the Periodontain Gene--
A data base
containing the unfinished P. gingivalis W83 genome
(available from the National Center for Biotechnology Information (NCBI), Unfinished Microbial Genomes at http://www.ncbi.gov) was searched for the presence of nucleotide sequences corresponding to the
NH2-terminal amino acid sequences of both chains of
periodontain using the TBALSTN algorithm (31). The sequence of the
clone, gnl|TIGR|P. gingivalis_112, which contained the
translated sequence for both chains of periodontain, was retrieved from
The Institute for Genomic Research data base (http://www.tigr.org). The
gene encoding periodontain was identified using the NCBI open reading frame finder program (also found at NCBI) and the amino acid sequence, obtained by conceptual translation of the entire open reading frame,
was further used for homology screening performed with the NCBI BLAST
search tool.
Enzyme Purification--
Previous experience in purifying enzymes
from culture supernatants of P. gingivalis indicated that
ice-cold acetone precipitation as an initial step was successful in
separating active proteinases from the bulk of peptides and proteins
present in or released into the growth medium. Similarly, G-150 gel
filtration as an early step was also utilized (32, 33), both to resolve
proteins into rough molecular weight fractions and to remove the excess heme and phytoheme that coprecipitate during acetone treatment. Subsequent anion exchange chromatography (Mono Q) and gel filtration (TSK) allowed periodontain to be purified to near homogeneity (Fig.
1A). Our yield of 11%
corresponded to over 1 mg of pure protein per each 5 liters of starting
culture fluid (Table I).
Physical Properties--
TSK gel filtration of the pure enzyme
yielded a single protein peak that eluted with a molecular mass of
~75 kDa, based on a linear regression data analysis from standards
(data not shown). However, SDS-PAGE yielded two distinct bands at 55 and 20 kDa, suggesting the presence of a heterodimer (Fig.
1B). Amino-terminal sequence analysis of each of these
subunits yielded the sequences of TEGVPAEVHALMDNGHFANDPMR and
DEWKKIGSVSVK for the heavy (55 kDa) and light (20 kDa) chains,
respectively. Analysis of the single protein species isolated from a
nondenaturing gel (Fig. 1A) gave two new amino-terminal
sequences in equimolar quantities, which corresponded to those
described above, confirming that periodontain is a heterodimer. It
should also be noted that either heating of the sample and/or presence
of SDS in the gel buffer was sufficient to separate the heavy and light
chains on electrophoresis, suggesting that the native heterodimer was
stabilized by ionic-hydrophobic interactions rather than a disulfide
bridge or covalent bonds. Isoelectric focusing yielded a pI of 5.3 for
the native protein (data not shown), whereas gelatin zymography
indicated that the 55-kDa heavy chain contained the catalytic active
site, but the 20-kDa light chain was devoid of enzymatic activity (Fig.
2).
Stability--
Periodontain activity was detected over a broad pH
range of 6.0-9.0, with the optimum being 7.5-8.0. The enzyme was
stable at 37 °C overnight, and at 4 °C for several weeks, when
stored in the absence of cysteine. The presence of reducing agent
resulted in a 50% loss of activity at 37 °C overnight, presumably
because of autodigestion. Heating to 60 °C caused complete loss of
activity. Samples were routinely stored at Activation and Inhibition--
Periodontain was completely
inactive in the absence of reducing agents, whereas full activity was
achieved with either free cysteine, Enzyme Specificity--
Periodontain was originally identified as
a unique proteinase that was able to inactivate
We then investigated the activity of periodontain on protein and
peptide substrates to further elucidate cleavage specificity. However,
the insulin Structure of the Periodontain Gene--
The region encoding for
the amino-terminal sequence of the 55-kDa catalytic subunit of
periodontain was amplified by PCR using degenerate primers and P. gingivalis W50 DNA. The 69-base pair PCR product was cloned and
sequenced. Using this structural information, the gene encoding
periodontain was extracted from the unfinished microbial genome at NCBI
(Fig. 5) and found to encode an 843-amino acid protein with a calculated molecular mass of 93,127 Da. The predicted size of the translated protein is ~20 kDa larger than we
found experimentally by both gel filtration and SDS-PAGE. However, examination of the gene product reveals that proteolytic processing of
the translated protein at Arg-147 and Lys-629 would yield a prepropeptide, a heavy chain containing the active site, and a light
chain derived from the carboxyl-terminal part of the proprotein that
correspond exactly to our experimentally determined amino-terminal sequences for these subunits (Fig. 6).
Thus, the native protein would have a predicted molecular mass of
76,727 Da, composed of a 52,981-Da catalytic heavy chain and a
23,764-Da carboxyl-terminal light chain, with a calculated pI of 5.18. This is virtually in complete agreement with our experimental findings
of a native protein of 70-80 kDa by gel filtration, composed of a
catalytic subunit of 55 kDa and a noncatalytic subunit of 23 kDa by
SDS-PAGE, with a pI of 5.3. All of these data suggest a potential role
in the processing of the pro-form of periodontain by both gingipains R
and gingipain K, all of which are abundantly present in P. gingivalis.
Distribution of Periodontain--
Using the cloned 69-base pair
catalytic amino-terminal fragment as a probe, Southern blot analysis of
the W50 strain revealed a single hybridizing band with each of the
restriction enzyme digests (data not shown). Furthermore, an
The multiple trypsin-like proteolytic activities have a
significant contribution to the virulence of P. gingivalis,
for both invasion and host defense evasion. In support of this concept, deletion of the two genes (rgpA and rgpB)
encoding various forms of gingipains R has been shown to attenuate
in vitro virulence of the knockout strain (34). In addition,
the use of antibodies to gingipains R polypeptide chain-derived
fragments has shown in vivo protection against P. gingivalis infection in a mouse model system (35).
Despite the importance of these bacterial proteinases, the abundance of
host-derived metalloproteinases and serine proteinases, including
active HNE, in the gingival crevicular fluid of individuals with severe
periodontitis, is still believed to be the primary factor responsible
for much of the extracellular matrix destruction that occurs in this
disease (36). The concentration of HNE within the neutrophil is near
3.0 µM, and it is likely to be as high as 268 µM at sites of inflammation (37), such as those that occur during the development of another major connective tissue disease, pulmonary emphysema.
The regulating inhibitor of HNE is Periodontain was easily purified from P. gingivalis culture
fluids and was found to be a cysteine proteinase that is apparently produced as a heterodimer. This enzyme is similar to the other characterized cysteine proteinases from P. gingivalis in
that all are primarily secreted in strain HG66, yet are present on membranes and vesicles in all other strains. Each has a signal peptide
sequence, a long prepropeptide, a large catalytic domain (~50 kDa),
and an additional carboxyl-terminal extension (20-40 kDa). In
contrast, whereas the gingipains have a highly restrictive specificity,
periodontain is characterized by an apparently nonspecific proteolysis
of peptides or denatured proteins. Although the ability of this enzyme
to hydrolyze From analysis of the amino terminus of the catalytic subunit and
subsequent cloning of this fragment, we were able to elucidate the
entire gene sequence from the partially completed genome sequence for
P. gingivalis obtained from NCBI. Searching the data base of
known sequences has revealed that periodontain is highly homologous to
both PrtT, a putative cysteine proteinase from P. gingivalis, and streptopain (EC 3.4.22.10), a secreted cysteine
proteinase from Streptococcus pyogenes (Table
III and Fig. 5). The prtT
gene, which has recently been cloned and sequenced (44, 45), is believed to encode a putative proteinase of 96-99 kDa. It is not surprising that periodontain and prtT have highly homologous
sequences, being from the same organism. Indeed, rgpA and
rgpB are two completely separate genes that code for almost
identical proteins with significantly overlapping specificity. However,
until the gene product for prtT has been purified and
characterized, functional similarities between PrtT and periodontain
cannot be assessed.
1-proteinase inhibitor, the primary endogenous
regulator of human neutrophil elastase. This new enzyme, referred to as
periodontain, belongs to the cysteine proteinase family based on
inhibition studies and exists as a 75-kDa heterodimer. Furthermore,
periodontain shares significant homology to streptopain, a proteinase
from Streptococcus pyogenes, and prtT, a
putative proteinase from P. gingivalis. Clearly, the
presence of this enzyme, which rapidly inactivates
1-proteinase inhibitor, could result in elevated levels
of human neutrophil elastase clinically detected in periodontal disease
and should be considered as a potential virulence factor for P. gingivalis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-proteinase inhibitor
(
1-PI) have been detected in GCF samples from patients with severe periodontal disease (22); however, despite the presence of
this inhibitor, there also remains a high HNE activity (23, 24),
indicating that the former must be present in either complexed, oxidized, or proteolytically inactivated forms. This is supported by
evidence showing that less than 35% of available
1-PI
in the GCF is active as an inhibitor (25). Furthermore, it has been shown that individuals with
1-PI deficiencies have a
significantly higher frequency of periodontal pocket depths
5 mm,
thereby predisposing them to manifestations of periodontal disease
(26).
1-PI (27). This enzyme is believed to be at least
partially responsible for the altered balance between the levels of HNE
and functional inhibitor in the GCF. In this report, we describe the
purification and properties of this enzyme, which we refer to as
periodontain, not only because of its function as a cysteine proteinase
but also because it may act as a putative factor in the dysregulation of
1-PI function in the periodontal cavity. In addition,
we provide the deduced protein sequence of periodontain as determined
by both partial peptide sequencing of the purified protein and analysis of the P. gingivalis genome.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-PI Inactivation Assay--
Detection of the
proteolytic activity of periodontain was determined by following its
ability to specifically inactivate
1-PI, using
-chymotrypsin as a target proteinase exactly as described previously
(27). Briefly, native
1-PI (0.15 nmol) was mixed with
samples containing a putative inactivating activity in assay buffer (50 mM Tris, 10 mM cysteine, pH 7.8) and allowed to
incubate for a desired time, after which an equimolar amount of
-chymotrypsin (0.15 nmol) was added to complex any remaining
functional
1-PI. The chymotrypsin substrate,
N-Suc-Ala-Ala-Pro-Phe-para-nitroanilide, was
added and, after a 4-min incubation, an end point absorbance at 405 nm
was read in a Molecular Devices SpectraMax Plus spectrophotometer in
order to determine the percentage of
1-PI remaining,
relative to controls.
10 °C. The protein pellet was redissolved in
20 mM Bis-Tris, 150 mM NaCl, 0.02%
NaN3, pH 6.8 (Buffer A), supplemented with 1.5 mM 4,4'-dithiopyridine disulfide, and dialyzed overnight
against the same buffer in a 10-kDa molecular mass cut-off membrane
(Spectra-Por) with two additional changes of Buffer A supplemented with
5 mM CaCl2. The dialyzed fraction was clarified
by centrifugation (40,000 × g for 30 min),
concentrated by ultrafiltration (Amicon PM-10 membrane), and applied in
20-ml fractions, each representing 5 liters of starting supernatant, to
a Sephadex G-150 column (5 × 105 cm) equilibrated with Buffer A,
at a flow rate of 30 ml/h. The activity was pooled, dialyzed against 50 mM Tris, pH 7.4 (Buffer B), and further purified by ion
exchange chromatography on a Mono-Q column (Amersham Pharmacia Biotech,
fast protein liquid chromatography system), with elution in a linear
gradient of 0-500 mM NaCl in Buffer B. Activity was
concentrated and final purification obtained by separation on a TSK-GEL
G3000SW (TosoHaas) column using 50 mM Tris, 200 mM NaCl, pH 7.4.
1-PI
after enzymatic inactivation employed the use of matrix-assisted laser
desorption ionization, with mass spectra acquired using a Vestec
matrix-assisted laser desorption ionization linear time-of-flight mass
spectrometer (Perspective Biosystems) at the Mass Spectroscopy Facility
(University of Georgia, Athens, GA) according to the manufacturer's instructions.
1-PI in our standardized assay (27) after
exactly 1 h of incubation. Representatives of the various classes
of proteinase inhibitors, at indicated concentrations, were
preincubated with enzyme for 5 min prior to the addition of
1-PI. The cleavage of inhibitor (%) was normalized to a
native inhibitor control in order to give the relative percent
inactivation for each compound or protein tested against periodontain.
The compound,
L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane (E-64), which stoichiometrically inhibits cysteine proteinases of the
papain family, was used to titrate periodontain. This allowed us to
quantitate stock solutions of periodontain in terms of active enzyme
and make dilutions to the desired concentrations for the degradation
experiments described below.
1-PI, inhibitor (20 µg) was incubated with 1 µg of periodontain for 4 h, after which the sample was subjected to 16%
SDS-PAGE to separate the ~ 3-kDa fragment obtained by cleavage within the loop. The fragment was analyzed for both amino-terminal sequence and molecular mass using an automated protein sequencer and
mass spectroscopy, respectively, as described above.
-chain, periodontain (8 nM) was incubated with this peptide substrate (40 µM) in assay buffer in a final
volume of 90 µl for desired time intervals. After stopping the
reaction by the addition of 10 µl of 10 N HCl, samples
were centrifuged (10,000 × g for 2 min), and the
entire supernatant (100 µl) was subjected to reverse-phase high
pressure liquid chromatography. Sample application to a Beckman
Ultrasphere 5 µm ODS column (4.6 × 250 mm) equipped with an
Ultrasphere 5 µm ODS guard column (4.6 × 45 mm) was carried out
in 0.1% trifluoroacetic acid in water (solvent A) and separations were
performed with a linear gradient of 0.08% trifluoroacetic acid in 80%
acetonitrile/water (solvent B) over 40 min at a flow rate of 1 ml/min.
The peptide elution was monitored at 215 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Periodontain is a heterodimer as a native
protein. A, nondenaturing PAGE of 1 µg pure
periodontain (lane 1) and 5 µg of albumin (lane
2). Note that albumin forms dimers and, to a lesser extent,
trimers when subjected to electrophoresis under nondenaturing
conditions. B, 10% SDS-PAGE of 1 µg of pure periodontain
indicating the presence of heavy and light chains.
Purification of P. gingivalis periodontain
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Fig. 2.
Periodontain heavy chain contains the
catalytic active site. Gelatin zymography of the pure enzyme was
preformed as described under "Experimental Procedures." Zones of
enzymatic activity are indicated by negative staining. The four
left lanes contain a serial dilution of purified periodontain
(1-0.125 µg), and the four right lanes contain identical
dilutions of enzyme preincubated with 100 µM E-64 for 5 min prior to electrophoresis.
80 °C for several
months with less than a 10% loss in activity.
-mercaptoethanol,
dithiothreitol, or dithioerythritol at 0.1 mM
concentration. Unlike the gingipains, which have higher activity in
free cysteine, no single reducing agent was superior in activating
periodontain. Furthermore, increasing concentrations of these reagents
(up to 10 mM) did not cause any additional stimulation of
periodontain activity. Finally, Ca2+ did not have a
stabilizing effect, and glycyl-glycine did not stimulate activity,
indicating additional differences between periodontain and the
gingipains (data not shown). Based on its requirement for a reducing
environment to become active, periodontain can be classified as a
cysteine proteinase, and this is confirmed by the fact that it is
readily inhibited by common cysteine proteinase inhibitors (Table
II). The ability of E-64 to inhibit
periodontain suggests that this enzyme is more closely related to
members of the papain family than other cysteine proteinases of
P. gingivalis, which are either not inhibited (gingipain K)
or only weakly inhibited (Rgps) by this compound.
Inhibition profile of periodontain
1-PI
through proteolytic cleavage. From the 3-kDa size difference of cleaved
versus native
1-PI noted on SDS-PAGE (Fig.
3A), we speculated that
periodontain caused hydrolysis within the exposed carboxyl-terminal RSL
of this molecule. Sequencing of the peptide generated by incubation of
1-PI with periodontain indicated that cleavage took
place after the glutamic acid (P5), and to a lesser extent,
phenylalanine (P7), of the
1-PI RSL (Fig.
3B). However, screening numerous synthetic
para-nitroanalide substrates with either Glu or Phe
specificity in the analogous position yielded no detectable cleavage by
periodontain. Indeed, even when we used the synthetic substrate
Phe-Leu-Glu-para-nitroanilide, which mimics the
P7, P6, and P5 residues within the
RSL of
1-PI, no hydrolysis was detected, indicating that
a specific amino acid residue at the site of hydrolysis does not
dictate the specificity of this enzyme.
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Fig. 3.
Inactivation of
1-PI by periodontain.
A,
1-PI (2 µM) was incubated
with periodontain (2 nM) for 0, 15, 30, 45, and 60 min
before the reaction was stopped by the addition of SDS sample buffer.
Right lane, sample was preincubated with 100 µM E-64 for 5 min prior to 60 min of incubation with
1-PI. The samples were electrophoresed on a 12%
separating gel. B, samples were separated on a 16% peptide
gel to isolate the 3-kDa fragment produced by this cleavage. Both
amino-terminal amino acid sequence analysis and mass spectroscopy of
the isolated fragment revealed that the major cleavage site was after
the P5 glutamic acid (thick arrow), and a minor
cleavage site was after the P7 phenylalanine (thin
arrow), relative to the P1-P1'
methionine-serine bond that forms the "bait" region of the RSL and
is attacked by HNE at the position indicated by an
asterisk.
-chain was hydrolyzed to such an extent that individual
cleavages could not be mapped, even at low E:S molar ratios (1:5000).
Rather, the high pressure liquid chromatography analysis revealed no
less than 10 peptides were generated from this 30-amino acid
polypeptide within 15 min, and complete digestion occurred in 60 min
(data not shown). Next, we examined the activity of periodontain on
native proteins. However, the enzyme was unable to degrade azocasein,
casein, lysozyme, collagen, fibrin, plasminogen, and fibrinogen (data
not shown). In contrast, when lysozyme was reduced, carboxymethylated,
or maleylated, complete digestion was noted in less than 10 min (Fig.
4). These results, together with activity
detected on the gelatin zymograph, indicate that periodontain cleaves
denatured or easily accessible polypeptide chains, but it cannot cleave
whole proteins with defined secondary or tertiary structure,
1-PI being the exception.
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Fig. 4.
Effect of periodontain on lysozyme and
reduced, carboxymethylated, or maleylated lysozyme. Periodontain
(30 nM) was incubated with 30 µM of either
lysozyme or reduced, carboxymethylated, maleylated lysozyme
(lysozyme-RCM) for the indicated times, and the reaction was
stopped by SDS sample buffer and electrophoresed on a 12% gel.
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Fig. 5.
Sequence alignment of periodontain, PrtT, and
streptopain. The putative gene products of prtT and
periodontain, both deduced from P. gingivalis strain W83
genome, and streptopain, from S. pyogenes, were aligned
according to homology modeling (black boxes indicate
identity, and gray boxes indicate similarity). The putative
catalytic cysteine and histidine residues of streptopain are marked
( ). The single underlined residues indicate the obtained
amino-terminal sequence of the light chain of periodontain, whereas the
double underlined residues indicate the sequence obtained
from both amino-terminal sequencing of the heavy chain and DNA
sequencing of the 69 base pair long PCR product. Arrows
indicate the putative cleavage sites necessary to form the mature
periodontain heterodimer from the nascent polypeptide.
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Fig. 6.
Proposed processing for the maturation of
periodontain. Structure of the periodontain protein with proposed
processing sites based on amino-terminal sequencing of the purified
heterodimer.
1-PI inactivating activity was detected in all P. gingivalis strains tested. Interestingly, periodontain was most
frequently associated with the membrane and outer membrane vesicles in
strains 33277, W50, W12, and 381, despite the fact that it was soluble
in strain HG66 (Fig. 7). This is also in
agreement with the distribution of other P. gingivalis proteinases (32, 33).
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Fig. 7.
Distribution of periodontain activity in
various P. gingivalis strains. Periodontain
activity from the indicated strains was measured against
1-PI using the standard assay as described under
"Experimental Procedures." Cell-associated activities (open
bars), vesicle-associated activities (hatched bars),
and soluble activities (solid bars) were normalized to 100%
for each strain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-PI, a plasma
protein that forms a complex with this proteinase and is rapidly
removed from the circulation and degraded (38). The inhibitor, however, can itself be inactivated by either oxidation at its reactive site or
by proteolytic cleavage by nontarget proteinases within the RSL region
(27, 39), and it is believed that both mechanisms occur during the
development of emphysema. Certainly, the high levels of active HNE in
the GCF, despite the presence of
1-PI, would suggest
that parallel mechanisms for inhibitor inactivation may be also
occurring in periodontal disease (37). In this respect, it has been
reported that whole cells or culture supernatants from P. gingivalis are capable of proteolytically inactivating
1-PI (40, 41), although it is clear that this is not due to any of the gingipain R or K forms because the inhibitor contains no
basic residues within its RSL (42). Thus, another proteinase(s) must be
involved in this process, and it is likely that periodontain, the
enzyme described in this report, serves this purpose.
1-PI, a native protein, is contrary to our
premise of its inability to cleave proteins with defined structure,
this can be explained by the fact that the RSL is present in a
flexible, extended conformation protruding above the protein core (43)
and, as such, may mimic a denatured protein or peptide.
Homology of periodontain with streptopain and prtT
(identity/similarity)
Periodontain and PrtT, containing 843 and 840 amino acid residues, respectively, are more than twice the size of the streptopain (398 residues) (Fig. 5). The function of the light chain of periodontain is presently unknown; however, the carboxyl-terminal domain for the putative prtT gene product has recently been shown to be identical to the putative product for a hemin-regulated gene, hemR (46). Due to the high homology of the catalytic domains of all three genes, we believe that periodontain should be classified with prtT and streptopain as an additional member of the C10 family of cysteine proteinases, as recently outlined (47).
Because P. gingivalis is an asaccharolytic organism, it must
acquire both carbon and energy predominantly from proteinaceous sources. Recent evidence using radiolabeled substrates has shown that
although this organism is very efficient at taking up dipeptides, it is
incapable of transporting single amino acids (which may endogenously be
present in GCF) across bacterial cell membranes (48). This is supported
by the fact that despite a large body of research performed on
extracellular endopeptidases from P. gingivalis, no
aminopeptidases or carboxypeptidases have been described to date.
Obviously, the gingipains, which only have a specificity for either
Lys-X or Arg-X bonds, would be restricted in the ability to degrade
large proteins to the size of di- or tripeptides, which could then be
transported into the bacterium. Therefore, it is possible that a number
of broadly specific proteinases and peptidases, including periodontain,
may be physiologically important to P. gingivalis, not
necessarily as virulence factors, but rather for nutrient acquisition.
This may be accomplished by three pathways. First, because periodontain
is the only peptidase in P. gingivalis so far described that
possesses the ability to inactivate 1-PI, this may be a
mechanism for increasing the levels of HNE, a nonspecific host
proteinase that might be utilized in protein degradation. Second, in
combination with HNE, periodontain may augment the degradation of
peptides produced by the actions of the gingipains. Third, there is
evidence that P. gingivalis produces a prolyl dipeptidyl
peptidase (49), a prolyl tripeptidyl peptidase (50), and an
uncharacterized collagenase.2
This proteolytic milieu in the GCF could aid in the final production of
peptides capable of being taken up by P. gingivalis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DE09761 (to J. T.) and by State Committee of Scientific Research (Komitet Badan Naukowych, Poland) Grant 6 P204A 019 11 (to J. P.).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, Life Sciences Bldg., Athens, GA 30602. Tel.: 706-542-1334; Fax: 706-542-1738.
2 D. Nelson, J. Potempa, T. Kordula, and J. Travis, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
Rgp, arginine-specific gingipain;
1-PI,
1-proteinase inhibitor;
HNE, human neutrophil elastase;
GCF, gingival crevicular fluid;
RSL, reactive site loop;
E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain
reaction;
NCBI, National Center for Biotechnology Information.
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