From the Department of Biochemistry, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Porphyromonas gingivalis is a
Gram-negative anaerobic bacterial species implicated as an important
pathogen in the development of adult periodontitis. In our studies of
P. gingivalis and ways to protect against periodontal
disease, we have prepared the monoclonal antibody mAb-Pg-vc and its
recombinant antibody, which are capable of inhibiting the
hemagglutinating activity of P. gingivalis (Shibata, Y.,
Kurihara, K., Takiguchi, H., and Abiko, Y. (1998) Infect. Immun. 66, 2207-2212). To clarify the antigenically related
hemagglutinating domains, we attempted to determine the minimum motifs
responsible for P. gingivalis hemagglutinin. Initially, the
9-kilobase EcoRI fragment encoding the 130-kDa protein was
cloned from the P. gingivalis chromosome using mAb-Pg-vc.
Western blot analysis of nested deletion clones, the competition
experiments using synthetic peptides, and the binding assay of the
phage-displayed peptides using the mAb-Pg-vc allowed us to identify the
minimum motifs, PVQNLT. Furthermore, the presence of multi-gene family
coding for this epitope was confirmed via Southern blot
analysis and PCR using the primers complementary to the domain
corresponding to this epitope. It is suggested that the
hemagglutinin-associated motif may be PVQNLT and that the gene families
specifying this motif found in P. gingivalis chromosome
encode many hemagglutinin and/or hemagglutinin-related proteases.
It is recognized that the adherence of bacteria to host tissues is
a prerequisite for colonization and one of the causative factors of
bacterial pathogenesis. Porphyromonas gingivalis is a
Gram-negative anaerobic bacteria that is isolated primarily from
infectious periodontal pockets and considered to be the major pathogen
for adult periodontitis (1, 2). Colonization of this bacterium in
gingival tissues is critical in the pathogenic process of periodontal
disease resulting in tissue destruction. Therefore, a number of
molecules including fimbriae (3), potential molecular adhesins such as
hemagglutinins (4), and lipopolysaccharides (5) responsible for
colonization have been identified as the virulence factors.
On the other hand, the cysteine proteases from this pathogen have been
extensively investigated because these enzymes could play an important
role in tissue destruction, activating host proenzymes, neutralizing
host defense mechanisms, and providing essential amino acids for growth
as well (6). Some of the enzymes are specific for cleavage of peptide
bonds containing arginine (7-12), others are specific for lysine
residues (13, 14), whereas still others exhibit specificity for both
amino acid residues (15, 16). Moreover, it has been proposed that the
adherence of P. gingivalis to erythrocytes (13), fibrinogen
(17), fibronectin (18), collagenous substrata (19), and other bacteria
(20) is mediated, at least in part, by such proteases at the cell
surface. Several investigators have indicated interesting results for
the correlation of the protease and hemagglutinin activities in
P. gingivalis (8, 21-23): arginine-specific cysteine
protease (arginine-gingipain)-deficient mutants exhibited decreased
hemagglutinating activity, which suggests notable properties of this
protease (8, 23). Recent enzymatic and molecular cloning analysis
directly revealed that these proteases are composed of two distinct
domains: one for proteolytic activity and the other for
hemagglutinating activity (9, 10, 14, 16, 22, 24).
The presence of hemagglutinating activity on the P. gingivalis cell surface was first reported by Okuda and Takazoe
(25). Indeed, the specific genes for P. gingivalis
hemagglutinin, such as hagA, which have four repeat units of
the hemagglutinin domain without the protease activity, have been
isolated (26). It is reasonable to expect that some hemagglutinin
molecules or other hemagglutinin domains encoded by many protease genes
in P. gingivalis possess the ability to degrade a broad
range of host proteins. Additionally, protoheme is an absolute
requirement for the growth of P. gingivalis (27), and it is
probably derived from erythrocytes in the natural niche of the organism
(28). Thus, the hemagglutinin molecule may be particularly important
for the organism not only for the attachment to the gingival tissues
but also to agglutinate and lyse erythrocytes to survive in
vivo. Although it is now well known that genes coding for
hemagglutinins and other proteases may share the domain specifying
hemagglutinin and hence consisting of a multigene family, the minimum
motif responsible for hemagglutinating activity in this family has only
been speculated by some investigators (29, 30).
Our approach to protect from periodontal diseases is to develop a
passive immunization system whereby the colonization of P. gingivalis cells onto human host tissues could be blocked. Toward
this goal, we first prepared a monoclonal antibody (mAb-Pg-vc) and a
recombinant single chain variable fragment antibody that inhibited the
hemagglutinating activity of P. gingivalis (31). Using these
antibodies, P. gingivalis genomic library was screened, and
the gene coding for the 130-kDa protein was isolated. The present
investigation will describe the detailed properties of the 130-kDa
protein, and its possible role in agglutination with erythrocytes will
be discussed.
Bacteria Culture Conditions and Preparation of the Vesicle
Fraction--
P. gingivalis 381 was grown in Todd-Hewitt
broth (Difco Laboratories, Detroit, MI) supplemented with hemin (0.2 µg/ml) and vitamin K1 (5 µg/ml) in an anaerobic
atmosphere (80% N2, 10% H2, 10%
CO2) for 24-48 h. Escherichia coli XL-1 Blue
(Stratagene) was grown on LB (1% trypton, 0.5% yeast extract, 0.5%
NaCl). For plasmid selection, 60 mg/liter of ampicillin was added to LB
agar plates. Vesicles were isolated basically by the method of Grenier and Mayrand (32), with a slight modification as previously reported (33).
Cloning and DNA Sequencing of the Gene Coding for 130-kDa
Protein--
P. gingivalis 381 genomic DNA was prepared,
digested with EcoRI, and inserted into the EcoRI
site of
For forward DNA sequencing, pHEM6 was digested with ApaI and
EcoRI and incubated with exonucleases III and mung bean
nuclease. Nested deletion clones were prepared following
recircularization and transformation into E. coli cells. For
reverse DNA sequencing, the SmaI fragment of pHEM6 was
subcloned, and the nested deletion clones were constructed in the same way.
DNA sequencing was performed with the Taq Dyedeoxy system
(Applied Biosystems, Inc.) and analyzed on an ABI 373A DNA sequencer. Complete coverage of both strands and the contiguous linkage were achieved by Long Ranger gel solution (FMC Bioproducts) using the LI-COR
4000L infrared automated sequencer (ALOKA). The DNA sequence data were
analyzed using the DNASIS programs (TaKaRa) and searched for in the DNA
data bank. Sequence grade plasmid DNA was purified by the automatic DNA
isolation system PI-50 (KURABO).
Preparation of Antibodies--
Preparation of the rabbit
polyclonal antibody raised against P. gingivalis vesicles
and its monoclonal derivative, mAb-Pg-vc, has been described previously
(31), both of which neutralize the hemagglutinating activity of
P. gingivalis vesicles. Recombinant 130k-HMGD was subjected
to SDS-polyacrylamide gel electrophoresis, and the protein was purified
by excising the gel corresponding to the recombinant 130-kDa protein
band. A rabbit serum antibody against the recombinant 130k-HMGD was
prepared after three intramuscular injections of the purified protein.
Prior to usage, the rabbit serum was absorbed with sonicated E. coli cells to minimize nonspecific immunoreaction.
Phage-displayed Peptide Library--
The Ph.D.TM ligand
screening system (7 peptides) was used to identify the epitopes
recognized by mAb-Pg-vc. The sterile 65 × 15 mm polystyrene Petri
dishes were coated with 1.5 ml of 0.01 M NaHCO3
containing mAb-Pg-vc and stored overnight at 4 °C in humidified
container. After the antibody solution was poured off, blocking buffer
(0.1 M NaHCO3, pH 8.6, 5 mg/ml bovine serum
albumin, 0.02% NaN3) was added and incubated for 1 h
at 4 °C. Then these dishes were washed with TBST buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Tween
20) six times, and 2 × 1011 phage in 1 ml of TBST
buffer was added and rocked gently for 1 h at room temperature.
These dishes were vigorously washed 10 times with TBST, and the bound
phage was then eluted by lowering the pH with 1 ml of 0.2 M
glycine-HCl (pH 2.2) containing 1 mg/ml bovine serum albumin. After
neutralization with 150 µl of 1 M Tris-HCl (pH 9.1), the
phages were amplified by infecting with E. coli NM522.
Recovered phage particles were subjected to a repeated biopanning
procedure in the same manner. After the third round of biopanning, a
total of 10 plaques were selected, and the phages were amplified. To
obtain the single-strand phage DNA, each culture supernatant was added
to polyethylenglycol/NaCl, and the recovered pellets were incubated
with iodide buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 4 M NaI) to remove the phage protein.
For sequencing, the primer (5'-CCC TCA TAG TTA GCG TAA CG) was used. To
quantify the amounts of phages bound to the coated mAb-Pg-vc,
enzyme-linked immunosorbent assay was carried out using horseradish
peroxidase-conjugated anti-M13 antibody.
Inhibition of Hemagglutinating Activity by Synthetic
Peptides--
The hemagglutinating activity of the vesicle fraction of
P. gingivalis 381 was assayed with washed rabbit
erythrocytes in round-bottomed microtiter plates. 70 µl of vesicle
solution (0.625 µg/ml) and 20 µl of antibody solution were
transferred into microtiter wells and incubated for 30 min at room
temperature. Then, 100 µl of 2% rabbit erythrocyte was added and
incubated for 2 h at room temperature. The inhibition of
hemagglutinating activity induced by adding synthetic peptides was
carried out under the same conditions as mentioned above.
Cloning of Other Genes Coding for mAb-Pg-vc-reactive
Proteins--
To clone other genes coding for the epitope sites
recognized by mAb-Pg-vc, the primers F (5'-TCC AAT GAA TTT GCT CCT) and R (5'-ATT TTC GAA TGA TTC GGA) were designed. These two primers were
able to amplify the region corresponding to the base positions 1298-1447 in pHEM6. PCR1 was
carried out by employing Pfu turbo (Stratagene) DNA
polymerase and P. gingivalis genomic DNA as a template, and
a reaction was performed for 30 cycles (94 °C for 1 min, 55 °C
for 2 min, and 72 °C for 2 min). The PCR products were precipitated
with 0.1 M of LiCl, 20 µg of glycogen, and 70% ethanol,
ligated into the pCR-Script SK(+) vector (Stratagene), cloned, and then sequenced.
Southern Blot Hybridization--
P. gingivalis
genomic DNA was digested with endonucleases, and the fragments were
separated in a 0.8% agarose gel. After alkaline denaturation, DNA
fragments were transferred onto a Hybond N+ membrane
(Amersham). Southern blot analysis was carried out using ECL labeling
and indirect detection kits (Amersham) according to supplier's recommendation.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
For SDS-polyacrylamide gel electrophoresis, P. gingivalis whole cells, vesicles, and other recombinant samples
were solubilized in 0.1% SDS solution, and subjected to 12%
acrylamide-bisacrylamide gels as described by Laemmli (34). Western
blots were prepared by electroblotting samples onto nitrocellulose for
1.5 h at 6 V in a transfer buffer (50 mM Tris-glycine,
20% methanol) using a semi-dry transfer cell system (Bio-Rad).
Cloning of the Gene Coding for 130k-HMGD--
Cloning of the gene
coding for 130k-HMGD and isolation of chimeric plasmid, pHEM6, is
outlined under "Experimental Procedures." To examine the antigenic
properties of the recombinant protein produced by HEM6, Western blot
analysis was carried out. As shown in Fig.
1, both the rabbit polyclonal antibody
and mAb-Pg-vc recognized the recombinant protein whose molecular mass
was 130 kDa (A and B, lanes 3,
respectively). This indicated that the subcloned 4.6-kb SacI
fragment originating from the recombinant phage, HEM9, retained the
same gene that encodes a recombinant 130-kDa protein responsible for
hemagglutinin. The rabbit polyclonal antibody raised against the
130k-HMGD exhibited the superimposable Western blot profile (Fig.
1C) as that of mAb-Pg-vc (Fig. 1B), suggesting
that these two antibodies recognized the same epitopes of the
hemagglutinin-related molecules of P. gingivalis.
Sequence Analysis of the 4.6-kb SacI Fragment--
To fully
characterize the 130k-HMGD, the 4.6-kb SacI fragment was
completely sequenced. The GC composition of this fragment was 48%, a
typical value for this organism (data not shown). Fig. 2 (A and B) shows
the gene organization and both nucleotide and deduced
amino acid sequences, respectively. It was revealed that the 4.6-kb
SacI fragment contained two open reading frames (ORFs).
The upstream ORF contained no ATG initiation codon, which is preceded
by a functional ribosome-binding site, ACATT, in this bacterium (35).
This ORF did possess the TAA termination codon, suggesting that the
upstream ORF might be a 3' portion of a putative gene, and expressed
under the control of the plasmid-borne lacZ' gene. The
upstream ORF consisted of 1223 codons capable of coding for the
131.5-kDa protein, which is in good agreement with the experimentally
determined molecular mass of recombinant 130k-HMGD. Detailed
characterization of this ORF will be discussed later.
The downstream ORF was composed of 212 codons coding for the 24.4-kDa
protein. Following computer analysis, it was revealed that this gene
product exhibited a high homology with the transposase of
IS1126 from P. gingivalis W83 (36).
Identification of the mAb-Pg-vc Binding Site in
130k-HMGD--
During the course of the sequencing experiments, a
series of nested deletion clones were constructed. Using these clones, we effectively examined the region responsible for mAb-Pg-vc binding (Fig. 3). Western blot analysis of the
E. coli lysate harboring each of the deletion plasmids
permitted us to specify the antigenically active domains (data not
shown). mAb-Pg-vc recognized Asn1-Gly734,
Asn1-Arg602,
Asn1-Asn482, and
Asn1-Thr475. In addition to these clones,
E. coli lysate harboring a 5' portion of
EcoRI-BamHI fragment, which encompasses amino
acid residues Asn1-Ser432, was also analyzed.
No immunopositive signals were observed in this clone (data not shown).
These results suggested that the epitope might reside within amino acid
residues from Ser432 to Thr475. We designate
this region as antibody recognition site-I (ARS-I).
In 5' deletion clones, both Ile603 and Pro783
exhibited positive signals; however, no signals were observed in
Ser989, suggesting that an additional epitope might exist
in the C-terminal portion (783-988) of 130k-HMGD. This second epitope
region is referred to as ARS-II.
Inhibition of Vesicle-associated Hemagglutinating Activity with
Synthetic Peptides--
As mentioned above, two immunoreactive
regions, ARS-I and ARS-II, were identified in recombinant 130k-HMGD.
Comparison of the deduced amino acid sequences corresponding to these
regions revealed the presence of three nearly resembling amino acid
stretches, PVQNLT, LKWD(N)AP, and LS(N)ES(D)FEN. The first two are
within ARS-I, whereas the remainder is outside of immunoreactive region.
To confirm the exact epitope domain, a series of overlapping peptides,
encompassing the amino acid residues from Lys423 to
Thr473, were synthesized (Fig.
4A). The monoclonal antibody,
mAb-Pg-vc, recognized the peptide-437AP. Using these peptides,
inhibition of the vesicle-associated hemagglutinating activity was
examined. As shown in Fig. 4B, peptide-431EG (EGSNEFAPVQNL)
and peptide-437AP (APVQNLTGSSVG) indicated an inhibitory effect on the
vesicle-associated hemagglutinating activity. Although the other
peptides KVTLKWDAPNGT, APNGTPNPNPNP, and NPNPGTT showed only limited
inhibition, KVCKDVTVEGSN and SSVGQKVTLKWD had no effect. This strongly
suggests that the region around the APVQNL structure possesses the
ability to compete with vesicles for binding to erythrocytes.
Phage-displayed Epitope Mapping--
To confirm the epitope
structure recognized by mAb-Pg-vc, phage clones expressing random
hepta-residues were screened by biopanning as outlined under
"Experimental Procedures." As a result, a total of seven
independent phage clones were isolated. Predicted amino acid sequences
are: FPVSQEL, HPVGNTS, KPLTIDT, THGPLSP, KHPTYRQ, YKLNPTR, and YTIGPPS.
Among these, quantification of phages bound to coated mAb-Pg-vc by
enzyme-linked immunosorbent assay indicated that following four
hepta-peptides were immuno-positive;
FPVSQEL, HPVGNTS, KPLTIDT, and
YKLNPTR (data not shown). Comparison of these
peptide sequences with that of 130k-HMGD suggested that PVQNLT
(438-443) seemed to be the structure most responsible for the epitope
site of mAb-Pg-vc (Fig. 4C). This conclusion was supported by the results shown in Figs. 3 and 4 (A and
B).
Southern Blot Analysis of P. gingivalis Chromosome Specific for the
mAb-Pg-vc Epitope--
Both the monoclonal antibody mAb-Pg-vc and the
rabbit polyclonal antibody raised against 130k-HMGD recognized several
proteins (Fig. 1). This suggested that P. gingivalis might
possess gene families that encode for mAb-Pg-vc-reactive proteins.
Alternatively, it was possible to explain that these multiple bands
were degradation products of progenitor proteins. To confirm either
possibility, Southern blot analysis was carried out. The P. gingivalis chromosome was digested with EcoRI or
SacI, digested doubly with EcoRI and SacI, and transferred onto membrane. For a positive control,
the 9-kb EcoRI fragment from HEM9 was also used. These blots
were analyzed using the 4.6-kb SacI and the 150-bp (epi-150)
fragments as probes. The latter fragment corresponds to
Ser433-Asn482, which contained the proposed
epitope structure, PVQNLT. As shown in Fig.
5, both probes exhibited superimposable
blotting profiles. As expected, the 9- and 4.6-kb bands were visualized
in genomic EcoRI and EcoRI-SacI
digests. In addition to these bands, several DNA fragments, ranging
from 4.3 to 19.3 kb in size, could also be detected in genomic
SacI digest. These data suggested that the P. gingivalis chromosome might contain multiple discrete loci coding
for the mAb-Pg-vc epitope.
PCR Amplification of Genes Coding for Epitope-specific
Site--
Because Southern blot analysis suggested the presence of
multiple discrete loci coding for the mAb-Pg-vc recognizable epitope, we attempted to clone these genes using a PCR technique (Fig. 6). The two primers were designed so that
the 150-bp fragment (epi-150), which was used as the probe in the
Southern blot analysis, could be amplified when the 4.6-kb
SacI fragment was used as a template (Fig. 6C,
lanes 2 and 4). When the P. gingivalis
chromosome was used as a template, several discrete DNA fragments were
detected on the agarose gel (Fig. 6C, lane 1).
These PCR products were ligated with plasmid and transformed onto
E. coli cells. We randomly picked 48 white colonies, and all
inserts were sequenced. As a result, following five independent clones
were isolated with duplications (Fig. 6D): epi-6 (165 bp),
epi-11 (135 bp), epi-12 (150 bp), epi-41 (165 bp), and epi-22 (1.4 kb).
The deduced amino acid sequence of epi-12 is exactly the same as that
of 130k-HMGD and several other registered gene products with or without
protease activity (GenBankTM accession numbers PGTLAGEN,
PGU42210, and PGU75366; Swisprot accession number AF017059). In
the other four clones, no identical sequences were found in the
registered genes; however, each clone exhibited the following
similarity; epi-06 (PGPRPR1), epi-11 (PGU41807), epi-41 (PGPRPR1), and
epi-22 (PGU41807). Comparison of DNA sequences between each
clone and that of the computer-aided counterpart protein indicated the
presence of conservative (underlined) and nonconservative
base-changes or deletion (boxed). In epi-06, for example, DNA
sequence corresponding to underlined region
(PNPNP) in counterpart protein
PGPRPR1 is different from that of epi-06; however, translated amino
acids were conserved. In addition, boxed NP sequence found in epi-06
was missing in PGPRP1. That the similar nucleotide disturbances were
also observed in the remaining three clones rules out the
possibility of contamination of strains used in the present study.
P. gingivalis is a Gram-negative anaerobic bacterium
that has become recognized as a major pathogen for adult periodontitis. One characteristic aspect of this bacterium is the formation of vesicle
particles, budding from the parental P. gingivalis cell surface. One of the pathogenic properties resulted from released vesicles is the ability to agglutinate erythrocytes or
hemagglutination. Using vesicle particles as antigen, we prepared two
antibodies, anti-vesicle rabbit serum and mouse monoclonal antibody
mAb-Pg-vc (31), capable of inhibiting hemagglutinating activity. In the present study, one of the antibodies, mAb-Pg-vc, was employed to screen
P. gingivalis genomic library, and the 4.6-kb
EcoRI-SacI fragment encoding for 130-kDa protein
was successfully isolated. Complete nucleotide sequencing revealed that
this fragment contained two ORFs; the upstream ORF is a 3' portion of
putative gene and is responsible for 130k-HMGD, whereas the downstream
ORF specifies IS1126-like (30, 36). Using the purified
recombinant 130k-HMGD protein as the "landmark," we carried out
three experiments: (i) Western blot analysis of nested deletion clones,
(ii) competition experiments using synthetic peptides, and (iii)
binding assay of phage-displayed peptide library. As a result, a
possible amino acid stretch, PVQNLT, responsible for antigenicity was
identified. Southern blot analysis of P. gingivalis genome
with DNA fragment corresponded to this antigenic region as probe, the
presence of gene family coding for PVQNLT traits was suggested, and
further PCR experiment coupled with nucleotide sequencing confirmed
this. Curtis et al. (29) and Kelly et al. (37)
have reported that the synthetic polypeptide
GVSPKVCKDVTVEGSNEFAPVQNLT (residues 907-931 of PrpR1) was
recognized in the serum from a patient with periodontitis and that this
domain must be related to bacterial colonization. These information
gave authenticity to our results.
Unexpectedly, however, 130k-HMGD exhibited no prominent
hemagglutinating activity. This observation strongly suggests that vesicle-associated agglutination of erythrocytes consists of many steps. Attachment of vesicles onto the surface of erythrocyte may
trigger the molecular cascade toward the hemagglutination, and native
130k-HMGD is involved in one of early events in these steps. To confirm
this hypothesis, further experiments will be necessary.
Using the DNA sequence data obtained in the present study, we performed
a homology analysis of the registered genes. As a result, several genes
could be identified with high homology. Among these, a total of 14 genes possessing the hemagglutinin domains were chosen, and their
protein structures are shown in Fig. 7.
Based on the protein structure, these proteins could be classified into
five groups. The first group is prtK (W50), prtP (W12), and lysine-specific cysteine protease (W83), in which the hemagglutinin domains exhibit the highest homology (99.3, 99.0, and
98.8%, respectively) with that of 130k-HMGD. The second group (kgp and hagD from 381) is the homologue of the
lysine gingipain K in which the C-terminal portion of residues (amino
acids 890-1150) are different. The third group consists of
rgp-1 (H66), prtR (W50), and prpR1
(W50), whose hemagglutinin domain is shorter than that of the first two
groups. The fourth group is composed of prtH (W83),
prtRII (381), rgp2 (H66), and rgpB and
possesses only short hemagglutinin domain. The last protein is the HagA
(381), which contains four repeating hemagglutinin domains in a single
molecule. In a comparison of the protein structures of 130k-HMGD and
the PrtK, it appeared that the "native" gene coding for 130k-HMGD is a homologue of the prtK and that the 3' two-thirds of
this gene was cloned in the present study. We predict P. gingivalis strain 381 might retain this gene. Additional computer
analysis of the registered genes other than P. gingivalis
provided us the interesting hemagglutinin molecules (HA1 and HA2)
originating from influenza virus. Of these, HA1 contains PVQNLT
homologue residue, PLQNLT (38). This further supports the fact that
PVQNLT might contribute to the immunogenicity in 130k-HMGD.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
zapII. The constructed phage library was screened by
mAb-Pg-vc, and the initial phage clone, HEM9, was isolated. This clone
produced an immunoreactive 130-kDa protein (130-kDa hemagglutinating
domain; 130k-HMGD). To obtain the minimum coding region for a
130k-HMGD, phage HEM9 DNA was digested with SacI. DNA
fragments were subcloned into pBluescript II KS(+) vector (Stratagene),
and the mAb-Pg-vc-reactive E. coli transformant harboring
pHEM6 was isolated.
RESULTS
View larger version (46K):
[in a new window]
Fig. 1.
Hemagglutinin-associated immunochemical
properties of P. gingivalis whole cells, vesicles, and
130k-HMGD. P. gingivalis whole cells, vesicles, and
130k-HMGD (lanes 1-3, respectively) were separated on
SDS-polyacrylamide gel electrophoresis. Transferred proteins were
analyzed with anti-vesicle rabbit serum, mAb-Pg-vc, and rabbit
anti-130k-HMGD (A-C, respectively).
View larger version (57K):
[in a new window]
View larger version (72K):
[in a new window]
Fig. 2.
The gene organization (A)
and nucleotide and deduced amino acid sequences (B) of
the 4.6-kb EcoRI-SacI fragment.
In B, the two potential epitope sites, ARS-I and ARS-II,
recognized by mAb-Pg-vc are boxed. The proposed
hemoglobin-binding domain (39) is underlined.
View larger version (17K):
[in a new window]
Fig. 3.
Identification of the mAb-Pg-vc binding site
in 130k-HMGD. The horizontal line at the top
represents the amino acid residues of 130k-HMGD. The bold
horizontal lines show the peptide domains expressed in each
deletion clone. Presence (+) or absence ( ) of immunoreactivity was
scored based on the Western blot analysis using a series of nested
deletion clones (data not shown).
View larger version (32K):
[in a new window]
Fig. 4.
Inhibition of vesicle-associated
hemagglutinating activity with synthetic peptides. A,
immunoreactivity of the synthetic peptides by mAb-Pg-vc. In this
experiment, a total of seven synthetic peptides were prepared. To
exemplify the relative position of these peptides, two amino acid
sequences (419Gly to 470Trp and
873Gly to 933Trp) corresponding to ARS-I
(boxed sequences) and a portion of ARS-II, respectively,
were shown at the top and bottom of seven
peptides. The vertical dashed lines denote the common
stretches present in both ARSs. 50 µl of the synthetic peptide (0.2 mM) were slot-blotted onto the nitrocellulose membrane. The
boxed sequence indicates the ARS-I site. B,
inhibition of hemagglutination by synthetic peptides spanning residues
423-475 of 130k-HMGD. Synthetic peptides (12-mers overlapping by 5-6
residues) were assayed for inhibition of hemagglutination of rabbit
erythrocytes mediated by purified vesicle fraction from P. gingivalis. E, erythrocytes; V, vesicles;
A, antibody (mAb-Pg-vc); P, peptide (0.1 mM). C, comparison of the putative epitope
residue of mAb-Pg-vc from the phage-displayed peptide library with the
residue of A.
View larger version (31K):
[in a new window]
Fig. 5.
Southern blot hybridization analysis of
P. gingivalis chromosomal DNA with the epitope coding
DNA probe. P. gingivalis chromosomal DNA was digested
with EcoRI (E) or SacI (S)
or doubly digested with EcoRI and SacI
(ES). For positive control, phage HEM9 DNA was digested with
EcoRI. These restriction fragments were separated on a 0.8%
agarose gel, transferred onto membrane, and analyzed with the 4.6-kb
EcoRI-SacI fragment (pHEM6 insert) or epi-150 as
probes.
View larger version (39K):
[in a new window]
Fig. 6.
Evidence of multigenicity of epitope site in
P. gingivalis by PCR analysis. A,
diagrammatic representation of the PCR experiment. Both F and R primers
were designed so that when P. gingivalis genomic DNA, one of
deletion clones (base positions 1-1806), or 4.6-kb
EcoRI-SacI was used as template, production of
the 150-bp fragment could be expected. Because the additional region
responsible for epitope was present, another deletion clone (base
position 2349-4600) was also used. B, assessment of
nonspecific amplification. To avoid misunderstanding due to nonspecific
amplification, the following combinations of primer(s) were employed,
and PCR was carried out using P. gingivalis chromosome as
template: FR, normal PCR; FF, F primer only;
RR, R primer only. M, 100-base ladder for size
marker. C, comparison of PCR-amplified products from genomic
DNA (lane 1), 1-1806 (lane 2), 2349-4600
(lane 3), and 1-4600 (pHEM6; lane 4).
D, deduced amino acid sequences of the chromosome-borne PCR
products. For details, see "Results."
DISCUSSION
View larger version (34K):
[in a new window]
Fig. 7.
Comparison of primary structures of the
hemagglutinin-associated proteins from P. gingivalis. The primary structures of the 130k-HMGD and
computer-accessed following 14 proteins possessing hemagglutinating
activity were aligned: prtK (PGU75366, 99.3%),
prtP (U42210, 2214278A, 99.0%) (16), (AF017059, 98.8%),
lysine gingipain K (2307336A, 82.4%) (40), kgp (D83258,
2219327A, 85.6%) (14), hagD (PGU68468, 85.5%), RGP-1
(A55426, 65.8%) (10), prtR (2219212A, 65.5%) (11),
prpR1 (PGPRPR1, 65.1%) (9), prtH (PRTH-PORGI,
61.7%) (41), prtRII (D26470, CPGI-PORG-1, 36.7%) (12),
rgp2 (PGU85038, 47.6%), rgpB (2314276A, 47.6%),
and hagA (PGU41807) (26). Open rectangles in 14 proteins indicate the regions homologous to the 130k-HMGD where single
amino acid changes are shown by dots. Two epitope sites
(PVQNLT) are also shown. Dotted vertical lines denote the
positions of hemoglobin receptor domains (HGP-15) proposed by Nakayama
et al. (39).
Recently, Nakayama et al. (39) have reported that the hemoglobin receptor domain (HGP-15) was found in several P. gingivalis proteases including arginine-gingipain (rgp1), lysine-specific cysteine proteases (prtP, and kgp), and hemagglutinin (hagA), which was located next to the putative hemagglutinin region. Using a lysine-gingipain-deficient mutant in which the uptake of hemin was greatly retarded, Okamoto et al. (42) indicated the defect of hemoglobin adsorption and heme accumulation. Interestingly enough, the duplicating immunoreactive amino acid stretch, PVQNLT, identified in the present study could be found in both sides of the HGP-15 in 130k-HMGD and other hemagglutinin-associated proteins as well. This fact is consistent with the idea that prior to penetration into erythrocyte, two PVQNLT stretches positioned in both side of HGP-15 might facilitate the attachment of P. gingivalis onto the erythrocyte cell surface. Once erythrocytes are coagulated, HGP-15 may function under the aid of the proteolytic activity equipped with the adjacent domain to obtain heme molecules. This gene structure seems to be ideal in this bacterium for the growth in the periodontal pocket to obtain the heme molecules as an iron source.
Deduced amino acid sequence of 130k-HMGD, the identification of short motif present in this protein, and the multiplication process of this motif in P. gingivalis cells shed the light on the two arguments that can explain the pathogenicity of this bacterium. First, deduced amino acid sequence indicated that the PVQNLT stretch is followed by a PN repeat, which is composed of typical structure-braking residues, making immunoresponsible residues outside of the cell wall. In ARS-II, a proline-rich stretch (TTTPPPG), another structure-braking feature, did exist that may again facilitate localization of the PVQNLT toward the molecular surface position. Second, nucleotide sequencing of the 4.6-kb EcoRI-SacI fragment indicated the presence of IS1126-like next to the gene coding for 130k-HMGD. Because the insertion sequence has the ability to move on chromosome concomitant with a particular gene, it is likely that this IS sequence may help spread the gene coding for PVQNLT all around the P. gingivalis chromosome and that it contributes to make this bacterium a more virulent strain.
In the present study, we identified the amino acid stretch, PVQNLT,
responsible for hemagglutinating activity. Because the synthetic
peptide, APVQNLTGSSVG, exhibited the inhibitory activity toward
hemagglutinin and because these motifs are so specific and widely
distributed at the P. gingivalis cell surface, this information might provide useful tools to establish a passive immunization system to prevent periodontal disease in human.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants-in-Aid for Scientific Research and Research for the Frontier Science from The Ministry of Education, Science, Sports and Culture of Japan C2 10671780 and A1 10357020, by Fund for Comprehensive Research on Aging and Health from The Ministry of Public Welfare of Japan Grant 96A2303, and by the Suzuki Memorial Grant of Nihon University School of Dentistry at Matsudo Young Researchers Grant 98-2004.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB019363.
To whom correspondence should be addressed: Dept. of Biochemistry,
Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan. Tel.: 81-47-360-9328; Fax: 81-47-360-9329; E-mail: yabiko{at}mascat.nihon-u.ac.jp.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s); ORF, open reading frame; ARS, antibody recognition site; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|