Porphyromonas gingivalis DPP-7 Represents a Novel
Type of Dipeptidylpeptidase*
Agnieszka
Banbula
§,
Jane
Yen
,
Aneta
Oleksy
§,
Pawel
Mak§,
Marcin
Bugno
§,
James
Travis
, and
Jan
Potempa
§¶
From the
Department of Biochemistry and Molecular
Biology, University of Georgia, Athens, Georgia 30602 and the
§ Institute of Molecular Biology, Jagiellonian University,
34-120 Krakow, Poland
Received for publication, September 26, 2000, and in revised form, November 28, 2000
 |
ABSTRACT |
A novel dipeptidylpeptidase (DPP-7) was purified
from the membrane fraction of Porphyromonas gingivalis.
This enzyme, with an apparent molecular mass of 76 kDa, has the
specificity for both aliphatic and aromatic residues in the P1
position. Although it belongs to the serine class of peptidases, it
does not resemble other known dipeptidylpeptidases. Interestingly, the
amino acid sequence around the putative active site serine residue
shows significant similarity to the C-terminal region of the
Staphylococcus aureus V-8 endopeptidase. The genes encoding
homologues of DPP-7 were found in genomes of Xylella
fastidiosa, Shewanella putrefaciens, and
P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di-
and tripeptidases, serve nutritional functions by providing dipeptides
to this asaccharolytic bacterium.
 |
INTRODUCTION |
Porphyromonas gingivalis, an oral anaerobic bacterium,
has been implicated as a causative agent of adult type periodontitis. As an asaccharolytic organism, P. gingivalis is totally
dependent on external sources of peptides, which are necessary for its
growth and proliferation. To fulfill such a fastidious nutritional
requirement, this bacterium evolved a complex system of proteolytic
enzymes, which are now recognized as important virulence factors in the development of periodontal disease (1). The best known and well
characterized enzymes of this system are gingipains R and K, arginine-
and lysine-specific cysteine proteinases (2). Working in concert with
the proteinases periodontain (3), collagenases/gelatinases (4-6), prtT
(7), and Tpr (8) as well as host proteinases, this array of enzymes has
the potential to degrade proteins from both the periodontal ligamentum
and surrounding tissues. Their concerted action leads to the formation
of a large pool of oligopeptides, which can be further utilized by
P. gingivalis and other oral bacteria. However, P. gingivalis cannot transport poly- and oligopeptides into the cell,
although it has the ability to thrive on dipeptides as a sole source of
carbon. For this reason, we have focused our attention on a specialized
group of P. gingivalis peptidases capable of hydrolyzing
oligopeptides to di- and tripeptides, which can be subsequently
metabolized by this periodontopathogen. In our previous report (9), we
presented the purification, characterization, and cloning of prolyl
tripeptidylpeptidase A, an enzyme that liberates tripeptides
from the N-terminal regions of substrates containing proline
residues in the third position. DPP1
IV, an enzyme with similar specificity
but only dipeptidylpeptidase activity, has also been cloned
(10), purified, and characterized (11, 12). Together with a recently
described angiotensinogen-converting enzyme analogue (13), all of these
proteases can hydrolyze peptide bonds containing proline residues. In
addition, the P. gingivalis genome contains three further
putative genes encoding proteinases homologous with dipeptidyl
peptidase IV, although their activities have not yet been identified
(9).
In the present study, we show purification, biochemical
characterization, and the gene sequence of a new cell
surface-associated serine protease with dipeptidylpeptidase activity.
This enzyme liberates dipeptides from the free amino terminus and has a
broad specificity for both aliphatic and aromatic residues in the
penultimate position.
 |
EXPERIMENTAL PROCEDURES |
Source and Cultivation of Bacteria--
P. gingivalis
DPP-7 was purified from strain HG66, a kind gift of Dr. Roland Arnold
(University of North Carolina, Chapel Hill, NC). The cells were grown
as described previously (14).
Protein Determination--
Protein concentration was determined
with the BCA reagent kit (Sigma), using bovine serum albumin as a standard.
Localization of Dipeptidylpeptidase Activity--
The
localization of active enzyme was checked in bacterial cells that had
been subjected to a previously described fractionation procedure (12).
All fractions, as well as the full culture, culture medium, and full
culture after sonication, were assayed for amidolytic activity against
H-Ala-Phe-pNA.
Enzyme Purification--
All purification steps were performed
at 4 °C except for FPLC separations, which were carried out at room
temperature. The cells were collected by centrifugation (6000 × g, 30 min) and resuspended in 50 mM potassium
phosphate buffer, pH 7.4. The outer membrane proteins were solubilized
with 0.05% Triton X-100. After 2 h of gentle stirring, unbroken
cells were removed by centrifugation (28,000 × g, 60 min). Proteins from the supernatant were precipitated with cold acetone
(60% final concentration), collected by centrifugation, and
redissolved in 50 mM potassium phosphate buffer, pH 7.0. After extensive dialysis against the same buffer, the sample was loaded onto a hydroxyapatite column (Bio-Rad) previously equilibrated with 20 mM potassium phosphate, pH 7.0, at a flow rate of 20 ml/h. The column was then washed until the A280 fell
to 0. Bound proteins were eluted with a potassium phosphate gradient
(20-300 mM), and fractions (7 ml) were analyzed for
amidolytic activity against H-Ala-Phe-pNA. The active
fractions were saturated with 1 M ammonium sulfate and
loaded onto a phenyl-Sepharose HP column (Amersham Pharmacia Biotech)
equilibrated with 50 mM potassium phosphate, pH 7.0, containing 1 M ammonium sulfate. The column was washed with
two volumes of the equilibration buffer, followed by a wash with buffer
containing 0.4 M ammonium sulfate and developed with a
descending gradient of ammonium sulfate from 0.4 to 0 M.
Active fractions were pooled, extensively dialyzed against 20 mM MES, pH 6.6, and applied onto a MonoS HR 5/5 FPLC
(Amersham Pharmacia Biotech) column equilibrated with the same buffer.
Bound proteins were eluted with a 0-300 mM NaCl gradient.
This allowed us to obtain a homogenous preparation of active proteinase.
Electrophoretic Techniques--
The SDS-PAGE system of Schagger
and von Jagow (15) was used to monitor enzyme purification and estimate
the enzyme molecular mass. For amino-terminal sequence analysis,
proteins resolved in SDS-PAGE were electroblotted onto polyvinylidene
difluoride membranes using 10 mM CAPS, pH 11, 10% methanol
(16). After staining 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.
Kinetic Analysis--
Routinely, the dipeptidylpeptidase
amidolytic activity was measured with H-Ala-Phe-pNA (1 mM) in 0.2 M HEPES, pH 7.8, at 37 °C. The
reaction was followed for specific time intervals in a thermostated
enzyme-linked immunosorbent assay reader (SpectraMax, Applied
Biosystem), and the release of p-nitroaniline was monitored at 405 nm. Other p-nitroanilide substrates were used in the
same manner. For inhibition studies, the enzyme was first preincubated with an inhibitor for 15 min at 37 °C, substrate was added, and residual activity was recorded. The initial steady-state velocity (v0) was determined by continuous assay for the
range of substrate concentrations (100 nM to 1 mM). Km and Vmax
were determined by hyperbolic regression of the kinetic data using the
software package Hyper Version 1.02 obtained from Dr. J. S. Easterby (University of Liverpool, United Kingdom).
Enzyme Fragmentation--
The purified dipeptidylpeptidase was
subjected to in-gel tryptic digestion
(17). Peptides were extracted and separated by microbore reverse-phase
high pressure liquid chromatography. Fractions absorbing at 210 nm were
manually collected, and their masses were determined by reflectron
matrix-assisted laser desorption ionization-time-of-flight mass
spectrometry using a Bruker Daltonics ProFlex instrument as described
previously (18). Selected peptides were subjected to Edman degradation
in a model Procise-cLS sequencer (PE Biosystems).
Identification of the DPP-7 Gene--
An unfinished P. gingivalis W83 genome data base, available from the Institute for
Genomic Research,2 was
searched for the presence of nucleotide sequences corresponding to the
amino-terminal and the internal DPP-7 amino acid sequences using the
TBLASTN algorithm (19). An identified contig
gnl[vert]TIGR[vert]P. gingivalis_1208 was retrieved from
the Institute for Genomic Research data base. The position of the DPP-7
gene was localized using the National Center for Biotechnology
Information (NCBI) 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--
The determination of substrate
specificity was based on the separation of the products of peptide
hydrolysis by reverse-phase chromatography. Peptides were first
incubated with 1 µg of DPP-7 at an enzyme/substrate molar ratio of
1:100 for 3 or 24 h in 50 µl of 200 mM HEPES, 100 mM NaCl, pH 8.0, 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
Supelcosil LC 18 column (Supelco) with an acetonitrile gradient of
0-60% in 0.075% trifluoroacetic acid in 50 min. Each peak, detected
at 210 nm, was collected, lyophilized, redissolved in 50% (v/v)
methanol plus 0.1% acetic acid, and subjected to analysis by mass spectrometry.
 |
RESULTS |
A 76-kDa dipeptidylpeptidase associated with P. gingivalis membranes was solubilized by mild detergent treatment.
This procedure released more than 90% of the amidolytic activity
against H-Ala-Phe-pNA into the medium. After acetone
precipitation and subsequent chromatography steps including the use of
hydroxyapatite, phenyl-Sepharose, and MonoS columns (Fig.
1), a pure enzyme preparation was
obtained. The homogeneity of the preparation and molecular mass of the
protein were checked both by SDS-PAGE (Fig.
2) and gel filtration on a TSK G3000
SW column (data not shown).

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Fig. 1.
Purification of P. gingivalis
DPP-7. Purification of DPP-7 from the acetone
precipitate of the P. gingivalis cell extract is
shown. Absorbance at 280 nm ( ) and amidolytic activity against
Ala-Phe-pNA ( ) are shown. a, separation
of DPP-7 on hydroxyapatite (100 ml) equilibrated with 20 mM
potassium phosphate buffer, pH 7.0; b, separation of DPP-7
obtained from the previous step on phenyl-Sepharose HP (25 ml)
equilibrated with 50 mM potassium phosphate, 1 M ammonium sulfate, pH 7.0, at a flow rate of 30 ml/h;
c, separation of DPP-7 on a MonoS FPLC column.
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Fig. 2.
SDS-PAGE of fractions obtained during the
purification of P. gingivalis DPP-7. 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, Triton X-100 extract of P. gingivalis; lane
c, acetone precipitate from Triton X-100 extract of P. gingivalis; lane d, hydroxyapatite column eluate;
lane e, phenyl-Sepharose column eluate; lane f,
MonoS column eluate.
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Inhibition Profile--
Based on the inhibition studies (Table
I) DPP-7 was classified as a serine
protease, being inactivated by diisopropylfluorophosphate, Pefablock,
and 3,4-dichloisocoumarin but not by typical cysteine class inhibitors
such as E-64 or iododoacetic acid. Metal chelators including EDTA and
1,10-orthophenanthroline, as well as reducing agents did not influence
its activity. The enzyme was not sensitive to inactivation by either
detergents (0.5% SDS, 1% Triton X-100) or heavy metal ions including
Zn2+, Co2+, and Ni2+. Human plasma
inhibitors, such as
1-proteinase inhibitor,
1-antichymotrypsin, and
2-macroglobulin,
did not affect enzyme activity, nor were they cleaved by DPP-7 (data
not shown).
pH Optimum and Stability--
Purified DPP-7 was active against
H-Ala-Phe-pNA over a broad pH range, from neutral to basic
pH (6.5-9.0) (Fig. 3). This activity also changed with the ionic strength of the buffer, reaching 200% at
0.5 M NaCl concentration in 100 mM HEPES, pH
8.0. DPP-7 was stable in 0.2 M HEPES, pH 8.0, for 1 week at
4 °C. The protease showed no appreciable loss of activity when kept
frozen at
80 °C for 1 month. After a 3-h incubation at either room
temperature or 37 °C, activity was reduced to 62 and 20%,
respectively. The optimum temperature for the hydrolysis of
H-Ala-Phe-pNA was determined to be 43 °C.

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Fig. 3.
pH optimum of the DPP-7 activity against
Ala-Phe-pNA. Enzyme activity was tested on
Ala-Phe-pNA substrate in different buffers including HEPES
( ), PIPES ( ), potassium phosphate ( ), Tris ( ), and MES
( ).
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Substrate Specificity--
Among the several chromogenic
substrates tested, only those with aliphatic or aromatic side chains
residues in the second, penultimate
position were rapidly hydrolyzed by DPP-7 (Table II). To further
confirm specificity, several synthetic peptides were also tested as
substrates for this enzyme. Again, only those with an aliphatic or
aromatic residue in the second position from the amino-terminal end
were cleaved (Table III), with glycine,
proline, or charged amino acids being not acceptable in the P1
position. The protease did not show any endopeptidase activity on
gelatin, insulin
-chain, carboxymethylated lysozyme, and azocasein
or type I collagen (data not shown). Purified DPP-7 was devoid of any
aminopeptidase activity and did not cleave model substrates with
blocked amino termini.
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Table II
Kinetic analysis for para-nitroanalide cleavage by DPP-7
Several other substrates, including H-Ala-Pro-pNA,
H-Ala-pNA, H-Gly-pNA, H-Ile-pNA,
H-Leu-pNA, H-Lys-pNA, H-Phe-pNA,
H-Gly-Arg-pNA, H-Gly-Glu-pNA,
H-Gly-Lys-pNA, H-Ala-Gly-pNA,
H-Gly-Gly-pNA, H-Ala-Ala-Phe-pNA,
H-Ala-Gly-Arg-pNA, H-Leu-Thr-Arg-pNA,
H-Ala-Phe-Pro-pNA,
N -benzoyl-DL-arginine-pNA,
N-Met-Ala-Pro-Val-pNA,
N-Suc-Ala-Ala-pNA,
N-Suc-Ala-Ala-Pro-Glu-pNA,
N-Suc-Ala-Ala-Pro-Leu-pNA,
N-Suc-Ala-Ala-Val-Ala-pNA,
Z-Ala-Ala-pNA, Z-Lys-pNA, Z-Arg-pNA,
Z-Glu-Glu-pNA, Z-Leu-Leu-Gly-pNA,
Z-Lys-Arg-pNA, Z-Phe-Arg-pNA,
Z-Phe-Val-Arg-pNA, Z-Tyr-Lys-Arg-pNA were tested,
but none of these was hydrolyzed by DPP-7.
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DPP-7 Sequence Analysis--
Purified DPP-7 was resolved on
SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane.
It had an amino-terminal sequence ADKGMMWLLNELNQENLDRMRELGFT. After
proteolytic in-gel digestion of the enzyme, additional internal
sequences were obtained, including DNKPYK, EMTYL, FAQFAN, VLPAML,
SVVPY, and LFFAGL. All of this sequence data allowed us to identify the
P. gingivalis genomic contig gln[vert]TIGR[vert]P.
gingivalis_ in the Unfinished Microbial Genomes data base, the
Institute of Genomic Research. An ORF corresponding to the DPP-7 amino
acid sequence was found, as indicated by the fact, that all sequences
of the DPP-7-derived peptides obtained by the enzyme polypeptide
fragmentation by trypsin were present in the protein primary structure
inferred from the nucleotide sequence of the ORF as shown in Fig.
4. The entire ORF corresponds to a
675-amino acid polypeptide with a calculated mass of 76247.4 Da.
Interestingly, the DPP-7 ORF contains the consensus sequence for the
active-site serine residue of serine type proteases,
TGGNSGSPVF. As indicated in Fig.
5, the DPP-7 carboxyl terminus exhibits a
high degree of identity to that of the V8 serine protease, particularly
around the putative active site serine residue. This is surprising,
since the P. gingivalis DPP-7 is a dipeptidyl peptidase
specific for small aliphatic and aromatic residues, whereas
Staphylococcus aureus V8 endopeptidase is specific toward
substrates containing glutamic and aspartic acid residues in the P1
position. The similarity search performed using the NCBI TBLASTN tool
against GenBankTM, EMBL, DDBJ, and PDB data bases showed no
significant similarity of DPP-7 to any other known dipeptidyl
peptidases, indicating that this enzyme could be regarded as a member
of a new family of proteases. Additional searches against data bases
containing unfinished and finished microbial genomes allowed us to
identify more genes coding for similar proteases with consensus active site sequence TGGNSGSPV (Fig. 6). A gene
of related protein has been found in the P. gingivalis W83
unfinished fragment of the complete genome between positions 1,360,759 and 1,362,718. The inferred primary structure of this putative
proteinase shows significant similarity to DPP-7 (267/691 identities).
Another organism, Shewanella putrefaciens, possesses two
related genes (gnl [vert]TIGR_24[vert]sputre 6401 and gnl
[vert]TIGR_24[vert]sputre 6410), while a plant pathogen, Xylella fastidiosa, contains one gene coding for similar
proteinase (gb[vert]AE004008.1[vert]).

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Fig. 4.
ORF for the gene coding P. gingivalis DPP-7. Underlined are sequences
obtained from the Edman degradation of the trypsin fragmented DPP-7
polypeptide chain. The putative active site serine residue is marked by
the black background.
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Fig. 5.
Comparison of the C-terminal regions of the
P. gingivalis DPP-7 (residues 664-695) and S. aureus V8 endopeptidase (residues 704-863).
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Fig. 6.
Multiple sequence alignment of P. gingivalis DPP-7 and its putative homologues. Sequences
of DPP-7-related proteinases were obtained from the conceptual
translation of the following ORFs retrieved from unfinished and
finished genome data bases: S1, S. putrefaciens
gnl [vert]TIGR_24[vert]sputre 6401; S2, S. putrefaciens gnl [vert]TIGR_24[vert]sputre 6410; X,
X. fastidiosa gb[vert]AE004008.1[vert]; P1,
P. gingivalis gnl [vert]TIGR[vert]P.
gingivalis_CPG.con; P2, P. gingivalis DPP-7
gnl [vert]TIGR[vert]P. gingivalis_CPG.con. The sequences
were subsequently aligned using the ClustalW multiple sequence
alignment tool.
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In addition, the computer-assisted search for sequential motifs
characteristic for transmembrane domains revealed the presence of two
such putative regions within the amino-terminal sequence of DPP-7, with
residues 7-24 and 62-78 most likely folded into hydrophobic
-helices responsible for membrane anchoring of this enzyme.
 |
DISCUSSION |
Several studies indicate that the outer membrane of P. gingivalis contains a complex proteolytic machinery, which serves
multiple physiological functions. In this study, we have described the identification of a novel proteinase localized on the bacterial surface.
The purified enzyme migrated as a single band with a molecular mass of
76 kDa on SDS-PAGE, and its amino-terminal sequence was located within
the primary structure of the translated product of the dpp-7
gene. Apparently, the enzyme is truncated at the amino terminus due to
the action of a lysine-specific proteinase, most likely gingipain K. Taking into account that the N terminus of DPP-7 contains membrane
anchorage domains, it is likely that the N-terminal truncation noted
here occurred during the isolation procedure and may not represent its
true membrane form.
The inhibition by typical serine protease inhibitors like
diisopropyl fluorophosphate, Pefablock, and phenylmethylsulfonyl fluoride, as well as resistance to sulfhydryl group blocking reagents and chelating agents, allowed us to classify this enzyme as a serine
protease. However, the P. gingivalis DPP-7 does not belong to any of the six previously described types of dipeptidylpeptidases (20). DPP I is a member of a cysteine class of peptidases and possesses
a broad specificity with an exclusion for basic amino acid and proline
residues in the P1 site of the scissile peptide bond (21). DPP VI is
another representative of the cysteine proteinase family with
dipeptidylpeptidase activity toward the broad spectrum of substrates
(22). DPP II, DPP IV, and DPP V belong to the S9 family of the serine
proteases (20). Both DPP II and DPP IV share similar specificity
directed against Pro and Ala residues in the penultimate position,
whereas DPP V is an enzyme secreted by Aspergillus fumigatus
with a unique substrate specificity limited to X-Ala,
His-Ser, and Ser-Tyr dipeptides (23). DPP III is also classified as a
serine peptidase, with its action being restricted to the Arg residue
in the P1 position (24). In terms of biochemical features, DPP-7
resembles a dipeptidyl aminopeptidase (DAP BII), which was isolated
from Pseudomonas sp. strain WO24, but the gene sequence of
that enzyme remains unknown and does not allow a sequence comparison of
these proteins (25). Because P. gingivalis
dipeptidylpeptidase does not exhibit any significant homology to any of
the dipeptidyl peptidases described so far, we have named this enzyme
DPP-7.
Interestingly, the P. gingivalis DPP-7 displays the
consensus sequence characteristic for the catalytic site of the V-8
like proteases, a group of endopeptidases cleaving after glutamic or aspartic acid residues (26). This region of significant sequence similarity is specifically located only at the C-terminal region of
both proteases and includes the putative active site serine residue.
Interestingly, we identified more genes coding for putative, DPP-7-related proteases in P. gingivalis, X. fastidiosa and S. putrefaciens. Based on the
enzymological and gene sequence data presented above, we conclude that
P. gingivalis DPP-7 does not belong to any of the peptidase
families previously reported and should, therefore, be regarded as a
prototype enzyme that defines a new family of dipeptidylpeptidases.
 |
ACKNOWLEDGEMENT |
Sequence data for DPP-7 was obtained
from the Institute for Genomic Research Web site.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DE 09761 and by Committee of Scientific Research (KBN, Poland) Grant 6PO4A 047 17.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. Tel.:
706-542-1713; Fax: 706-542-3719; E-mail: potempa@arches.uga.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008789200
2
Sequence data for DPP-7 was obtained from the
Institute for Genomic Research site on the World Wide Web.
 |
ABBREVIATIONS |
The abbreviations used are:
DPP, dipeptidylpeptidase;
MES, 4-morpholineethanesulfonic acid;
CAPS, 3-(cyclohexylamino)propanesulfonic acid;
pNA, p-nitroanilide;
Suc-, succinyl-;
Z-, benzyloxycarbonyl;
contig, group of overlapping clones;
ORF, open
reading frame.
 |
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