This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakai, E.
Right arrow Articles by Nakayama, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakai, E.
Right arrow Articles by Nakayama, K.

 Previous Article  |  Next Article 

Journal of Bacteriology, June 2007, p. 3977-3986, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01691-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Construction of Recombinant Hemagglutinin Derived from the Gingipain-Encoding Gene of Porphyromonas gingivalis, Identification of Its Target Protein on Erythrocytes, and Inhibition of Hemagglutination by an Interdomain Regional Peptide{triangledown}

Eiko Sakai,1 Mariko Naito,2 Keiko Sato,2 Hitoshi Hotokezaka,3 Tomoko Kadowaki,4 Arihide Kamaguchi,5 Kenji Yamamoto,4 Kuniaki Okamoto,1 and Koji Nakayama2*

Division of Oral Pathopharmacology, Department of Developmental and Reconstructive Medicine,1 Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology,2 Division of Orthodontics and Dentofacial Orthopedics, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan,3 Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan,4 Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, Sapporo, Japan5

Received 2 November 2006/ Accepted 13 March 2007


arrow
ABSTRACT
 
Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44720-1081, a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44720-1138, did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44720-1138 but not in HGP44720-1081, could bind HGP44720-1081 in a dose-dependent manner and effectively inhibited HGP44720-1081-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44720-1081-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44720-1081-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44720-1081 could bind to asialoglycophorin with a dissociation constant of 3.0 x 10–7 M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.


arrow
INTRODUCTION
 
Porphyromonas gingivalis, a gram-negative anaerobic bacterium, has been implicated as a major pathogen in the development and progression of chronic periodontitis. This bacterium expresses numerous potential virulence factors, including fimbriae (21), lipopolysaccharides (19), and various proteases and hemagglutinins (5). Since P. gingivalis is asaccharolytic and obtains nutrition for growth from amino acids and peptides in the local environment, attachment of adhesin molecules to oral epithelial cells and tissues is important for its survival. While it is well recognized that hemagglutination is related to the ability of bacteria to adhere to host tissues, which is an initial step in bacterial infection, P. gingivalis-mediated hemagglutination has special significance since this bacterium grows much faster in a culture with hemin than in a culture without hemin.

Previously, workers have attempted to identify the hemagglutinin molecule. Okuda and Takazoe first reported that bacterial surface components of a bacterium had hemagglutinating activity (37). Ogawa and Hamada reported that P. gingivalis 381 FimA fimbriae and their oligopeptide segments have activity that agglutinates erythrocytes (35), while other researchers found that purified FimA fimbriae from P. gingivalis 381 exhibited no hemagglutinating activity (61) and that removal of fimbriae from P. gingivalis W12 cells had no effect on the hemagglutinating activity (5). Hemagglutinins with apparent molecular masses of about 24,000, 37,000, and 44,000 Da were partially purified by Inoshita et al. (20) from a culture supernatant of P. gingivalis 381. Okuda et al. (38) also partially purified hemagglutinins and obtained similar results. They reported that the hemagglutinating activity is not inhibited by sugar but is inhibited by arginine and arginine-containing peptides. Other workers have also partially purified hemagglutinins (33) (17).

P. gingivalis produces an arginine-specific cysteine proteinase (Arg-gingipain, Rgp) and a lysine-specific proteinase (Lys-gingipain, Kgp) (44). Molecular genetic analyses have revealed that Rgp is encoded by two genes, rgpA and rgpB, and that Kgp is encoded by a single gene, kgp (31). The proteins encoded by rgpA and kgp have similar structures and consist of an N-terminal propeptide region, a proteolytic domain, and C-terminal adhesin domains (HGP15 [HbR], HGP17, HGP27, and HGP44). The adhesin domains are also encoded by the hemagglutinin-encoding gene hagA; these regions are quite similar to C-terminal regions (HGP44 and HbR) of the rgpA and kgp products (1, 16). Monoclonal antibodies (MAb) 61BG1.3 and Pg-vc that inhibited hemagglutination of P. gingivalis were found to recognize a peptide in an adhesin domain (HGP44 encoded by rgpA) encoded by rgpA, kgp, and hagA (4, 24, 47). We previously found that an rgpA rgpB mutant and an rgpA kgp mutant had decreased abilities to agglutinate erythrocytes (31) and that an rgpA kgp hagA triple mutant had no hemagglutinating activity (46), suggesting that all three genes are responsible for hemagglutination. It has been reported that Kgp proteinase-adhesin complexes have hemagglutinating activity (42), while a single-chain 50-kDa form of RgpA has no such activity (41), suggesting that the proteinase domain alone is not sufficient for hemagglutination. Although these findings indicate that adhesin domain proteins are the likeliest candidates for hemagglutinin, direct evidence that identifies the molecule that is actually responsible for hemagglutination and adhesion has not been obtained yet. In order to clarify this issue, we overexpressed and purified various recombinant adhesin domain proteins derived from the HGP44 region. In this study, we obtained the first evidence showing that fully processed HGP44 can agglutinate human erythrocytes without any other HGPs or gingipains, and we determined the target molecule on the membrane of human erythrocytes.


arrow
MATERIALS AND METHODS
 
Bacterial strains and culture conditions. Cells of P. gingivalis strains ATCC 33277 and KDP137 (rgpA kgp hagA) (46) were grown anaerobically (10% CO2, 10% H2, 80% N2) in enriched brain heart infusion (BHI) medium (31). Overnight cultures were centrifuged (10,000 x g, 10 min), washed with phosphate-buffered saline (PBS), and suspended in PBS. The bacterial suspensions were then diluted to obtain a twofold dilution series with PBS. A 100-µl aliquot of each suspension was mixed with an equal volume of a human erythrocyte suspension (1% erythrocytes in PBS) for the hemagglutination assay. The rgpA kgp double mutant KDP137 was grown anaerobically (10% CO2, 10% H2, 80% N2) in enriched BHI medium for 40 h. The culture supernatant was used for purification of RgpB proteinase.

Proteins and antibodies. Neuraminidase from Clostridium perfringens type V, chymotrypsin (C3142), human glycophorin A, human asialoglycophorin A, human transferrin, human albumin, fetuin and asialofetuin from fetal calf serum, mouse anti-human glycophorin A/B (G7650) MAb, and mouse anti-human glycophorin C (G7775) MAb were purchased from Sigma. Rabbit anti-human band 3 polyclonal antibody was obtained from Santa Cruz (Santa Cruz, CA). Mouse anti-human band 3 MAb was obtained from Abcam (Cambridge, MA). Mouse anti-human CD239 (lutheran) was purchased from Serotec (Kidlington, Oxford, United Kingdom). Rabbit anti-hemoglobin binding protein (HbR) antibody was prepared as previously described (32). Peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) and peroxidase-conjugated anti-mouse IgG were purchased from Dako. Blood of cows, horses, and guinea pigs was purchased from Cosmo Bio (Tokyo, Japan).

Preparation of anti-HGP44 antibody. Recombinant glutathione S-transferase (GST)-HGP44720-1138 (rGST-HGP44720-1138) fusion protein was obtained as described previously (23). rHGP44720-1138 protein was obtained by cleavage of the GST fusion protein with PreScission protease (Amersham Pharmacia Biotech, Piscataway, NJ) used according to the manufacturer's instructions. After cleavage of the GST-HGP44720-1138 fusion protein in a glutathione-Sepharose 4B column with PreScission protease, rHGP44720-1138 was eluted from the glutathione-Sepharose 4B column with elution buffer. The eluate was then applied to the glutathione-Sepharose 4B column again to remove contaminating GST and PreScission protease. The purity of rHGP44720-1138 was then determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). rHGP44720-1138 was mixed with Freund's complete adjuvant, and the mixtures were injected into mice (BALB/c and ICR) and rabbits (Japan White) subcutaneously using two booster shots of a mixture of antigen and Freund's incomplete adjuvant, which resulted in anti-HGP44 antisera. Animal care and experimental procedures were conducted in accordance with the Guidelines for Animal Experimentation of Nagasaki University and with approval of the Institutional Animal Care and Use Committee. Anti-HGP44 IgG was purified from antiserum obtained from rabbits using protein A-conjugated Sepharose 4B (Amersham Pharmacia Biotech).

Purification of His-tagged rHGP44. Primers were designed on the basis of the predicted sequences of genomic DNA coding for the HGP44 domain protein in the rgpA gene of P. gingivalis ATCC 33277. Truncated mutant HGP44 proteins were generated by removing codons by PCR mutagenesis. The sequences of the primers used were 5'-CATATGAGCGGTCAGGCCGAGATTGTTC-3' (forward primer) and 5'-CTCGAGGCGCTTGCCGTTGGCCTTGATC-3' (reverse primer) for HGP44A, 5'-CCATATGAGCGGTCAGGCCGAG-3' (forward primer) and 5'-GCTCGAGTGCCGTAATCGTCTCTTC-3' (reverse primer) for HGP44B, 5'-CCATATGATTTGGATTGCCGGACAAGGA-3' (forward primer) and 5'-GCTCGAGTGCCGTAATCGTCTCTTC-3' (reverse primer) for HGP44C, 5'-CCATATGGACGGCACGAAGATC-3' (forward primer) and 5'-GCTCGAGTGCCGTAATCGTCTCTTC-3' (reverse primer) for HGP44D, 5'-CCATATGGACGTTACGGTAGAAGGATCC-3' (forward primer) and 5'-GCTCGAGTGCCGTAATCGTCTCTTC-3' (reverse primer) for HGP44E, and 5'-CCATATGACGATCGATGCAGACGGTGACGGG-3' (forward primer) and 5'-GCTCGAGTGCCGTAATCGTCTCTTC-3' (reverse primer) for HGP44F. To verify their identities, all clones were subjected to DNA sequencing using plasmid templates and a dideoxy sequencing kit (Thermal Cycler sequencing kit; Amersham Pharmacia Biotech) with a Long Reader Sequencer 4200 (Li-Cor). The resulting fragments were then inserted into the NdeI-XhoI site of plasmid pET22b (Novagen), and the recombinant expression plasmid was then transformed into Escherichia coli BL21(DE3). Positive transformants were selected on Luria-Bertani agar plates containing carbenicillin (50 µg ml–1), and colonies were used for inoculation into Luria-Bertani broth for large-scale culture. Isopropyl-ß-D-thiogalactoside (IPTG) was added to the culture at a concentration of 0.1 µM, and this was followed by incubation for 2 h to overproduce the recombinant proteins. The recombinant proteins were purified by using the Ni-nitrilotriacetic acid purification system (Invitrogen, Carlsbad, CA). In this study, hexahistidine (His6)-tagged rHGP44 proteins were used unless indicated otherwise.

Hemagglutination assay. Human erythrocytes from type A, B, AB, and O blood were obtained from healthy human volunteers. The erythrocytes were sedimented by centrifugation, and the plasma and buffy coat were removed by aspiration. The cells were washed three times by centrifugation at 4°C in PBS. The hemagglutinating activity was determined in round-bottom microtiter plates. rHGP44 proteins were diluted to obtain a twofold dilution series in PBS, and then an equal volume of a 2% suspension of washed human erythrocytes was added. The mixtures were incubated at room temperature for 60 min. Human erythrocytes pretreated with neuraminidase (10 to 100 mU; Sigma) in PBS for 1 h at 37°C were also used to examine hemagglutination.

ELISA for binding of synthetic peptides to HGP44B. The interdomain regional peptide GVRSPEAIRGRIQGTWRQKT (Pep-1) corresponding to residues 1083 to 1102, peptide GVASPEAIRGAIQGTWAQKT (Pep-2) in which alanine was substituted for Arg1085, Arg1093, and Arg1099 of Pep-1, and peptide ADHFQYGQVIPSDTHTLWPN (Pep-3) corresponding to residues 745 to 764 (phenylalanine was substituted for Asp748 because of difficulty with peptide synthesis) were obtained from Sigma Genosis. The abilities of these peptides to bind to HGP44B were determined by an enzyme-linked immunosorbent assay (ELISA). An ELISA plate (SUMILON 96-well ELISA plate; Sumitomo Bakelite Co., Ltd. Tokyo, Japan) was coated for 2 h at 37°C with the peptides in carbonate buffer (pH 9.0). Nonspecific sites were blocked with 1% bovine serum albumin-PBS. After washing, the plates were incubated with rHGP44B in bovine serum albumin-PBS for 2 h at 37°C. Bound HGP44B was reacted with rabbit anti-HGP44 antibody and horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin and was subsequently developed with o-phenylendiamine dihydrochloride.

Binding of HGP44B to erythrocyte ghost membrane proteins. Human erythrocytes were washed three times with cold PBS, and the buffy coat was carefully removed with each wash. Hemoglobin-free erythrocyte membrane white ghosts were prepared by hypotonic lysis of the washed cells in 5 mM sodium phosphate buffer (pH 8.0), essentially using the procedure of Fairbanks et al. (11). Membrane proteins on SDS-PAGE gels were electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The blots were blocked with 5% skim milk for 1 h at room temperature, probed with 5 µg/ml of HGP44B overnight at 4°C, washed, incubated with anti-HGP44 antibody for 1 h at room temperature, washed, incubated with HRP-conjugated secondary antibodies, and finally detected with ECL-plus (Amersham Pharmacia Biotech).

Protein cross-linking and immunoprecipitation. Human erythrocytes were washed with cold PBS as described above. The washed cells (500 µl) were incubated with or without rHGP44B (200 µg) for 60 min at room temperature. Cells were then treated with the amino group-reactive homobifunctional cross-linking agent BS3 (Pierce) as follows. BS3 was added to rHGP44B-pretreated erythrocytes at a concentration of 2 mM, and cross-linking was allowed to proceed for 30 min at room temperature. The reaction was terminated by addition of Tris-HCl (0.1 M, pH 7.4) to a final concentration of 20 mM. The cells were washed three times with PBS to remove the residual rHGP44B and BS3. Erythrocyte membrane white ghosts were prepared as described above. The white ghost membrane was then solubilized with a solution containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Sigma). The membrane lysate was subjected to immunoprecipitation with rabbit anti-HGP44 antibody used according to the manufacturer's instructions. The resulting precipitate was subjected to 10% SDS-PAGE and immunoblot analysis.

SDS-PAGE and immunoblot analysis. SDS-PAGE was performed by using the method of Laemmli (25). The gels were stained with 0.1% Coomassie brilliant blue (CBB) R-250. For immunoblot analysis, proteins on SDS-PAGE gels were electroblotted onto a PVDF membrane. The blots were blocked with 5% skim milk for 1 h at room temperature, probed with an antibody against HGP44, glycophorin A/B, glycophorin C, band3, or lutheran overnight at 4°C, washed, incubated with HRP-conjugated secondary antibodies, and finally detected with ECL-plus (Amersham Pharmacia Biotech).

Purification of RgpB proteinase. Cell-free culture supernatant of KDP137 (rgpA kgp hagA) was obtained by centrifugation at 10,000 x g for 20 min at 4°C. Ammonium sulfate was added to the culture supernatant to obtain 75% saturation. The precipitated proteins were collected by centrifugation at 10,000 x g for 20 min and suspended in 10 mM sodium phosphate buffer (pH 7.0). After dialysis against 10 mM sodium phosphate buffer (pH 7.0) at 4°C overnight, the insoluble material was removed by centrifugation at 25,000 x g for 30 min. The remaining soluble fractions containing Rgp activity were applied to a DEAE-Sepharose CL6B column that had been equilibrated with the same buffer. After the column was washed with the same buffer, elution was performed with the buffer containing 50 and 100 mM NaCl sequentially. Fractions with proteolytic activity were eluted with 100 mM NaCl. The active fractions were purified further by Sephacryl S-100 gel filtration chromatography. The active fractions were pooled and concentrated. Enzymatic activity was assayed as described previously (22).

Surface plasmon resonance (BIACORE). The interaction between HGP44B and asialoglycophorin was analyzed with BIACORE X (BIACORE AB, Tokyo, Japan). Asialoglycophorin (100 µg ml–1) in 10 mM sodium acetate (pH 4.0) was immobilized on a CM5 carboxymethyl-dextran sensor chip using the amine-coupling method. HGP44B (10 µg ml–1 to 100 µg ml–1) in 10 mM HEPES (pH 7.4) containing 150 mM NaCl was passed over the surface of the sensor chip at a flow rate of 20 µl min–1. The interaction was monitored by determining changes in surface plasmon resonance response at 25°C. After 3 min of monitoring, the same buffer was introduced onto the sensor chip in place of the HGP44 solution to start the dissociation. Both the association rate constant (Ka) and the dissociation rate constant (Kd) were calculated by using the BIAevaluation software (BIACORE AB) and the program 1:1 (Langmuir) binding model. The dissociation constant (KD) was determined from Kd/Ka.


arrow
RESULTS
 
Recombinant HGP44B has hemagglutinating activity. Molecular genetic analyses have revealed that the rgpA, kgp, and hagA genes are responsible for hemagglutination (31, 46). Adhesin domain HGP44 is encoded by three genes designated rgpA, kgp, and hagA (Fig. 1A). To determine whether HGP44 is actually responsible for hemagglutination, we generated rHGP44. We initially constructed a GST-HGP44720-1138 fusion protein and purified HGP44720-1138 after proteolytic digestion (Fig. 1B), and we examined the ability of the purified protein to agglutinate human erythrocytes; however, unexpectedly, the protein was not able to agglutinate the erythrocytes (data not shown). Next, to determine whether the HGP44 region was responsible for P. gingivalis-mediated hemagglutination in our experimental system using strain ATCC 33277, we constructed an antibody against rHGP44720-1138 and investigated the effect of the anti-HGP44720-1138 antibody on P. gingivalis-mediated hemagglutination. As shown in Fig. 1C, anti-HGP44720-1138 antibody completely blocked P. gingivalis-mediated hemagglutination at concentrations of 2.5 to 10 µg per well. Hemagglutination was not blocked by a preimmune antibody or an anti-HbR antibody, showing the specificity of HGP44-dependent hemagglutination in our system. Veith et al. reported that the C-terminal end of the HGP44 domain protein on the cell surface of P. gingivalis was predicted to be at Ala1081 (58). Therefore, we constructed and purified a protein with a C-terminal deletion (HGP44B [HGP44720-1081]), proteins with an N-terminal deletion and a C-terminal deletion (HGP44C [HGP44820-1081], HGP44D [HGP44873-1081], HGP44E [HGP44915-1081], and HGP44F [HGP44982-1081]), and full-length HGP44 (HGP44A [HGP44720-1138]) using a His6-tagged recombinant protein construction and purification system, and we determined their abilities to agglutinate erythrocytes (Fig. 2A and B). HGP44E lacks residues 720 to 914 that contain FEED (residues 886 to 889). HGP44F lacks residues 720 to 980 that contain FEED (residues 886 to 889), PVQNLT (residues 926 to 931), and PNPNPNPNPNPNP (residues 950 to 962) (Fig. 1B). These amino acid sequences are thought to be responsible for binding to fibrinogen, fibronectin, and/or hemoglobin. As shown in Fig. 2C, HGP44B was able to agglutinate erythrocytes, whereas HGP44A, HGP44C, HGP44D, HGP44E, and HGP44F could not agglutinate erythrocytes.


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 1. P. gingivalis-mediated hemagglutination is inhibited by anti-HGP44 antibody. (A) Domain structures and homologies of gingipain adhesins RgpA, RgpB, Kgp, and HagA. RgpA and Kgp consist of signal peptide, propeptide, mature proteinase, and adhesin domain regions (HGP44, HbR, HGP17, and HGP27). RgpB has signal peptide, propeptide, and RgpB proteinase domain regions. HagA has a repeated structure consisting of HGP44-like domains and HbR. R and K indicate cleavage sites for Rgp and Kgp, respectively. (B) Amino acid sequence of HGP44 region derived from DNA sequences of rgpA. (C) Inhibition of P. gingivalis-mediated hemagglutination by anti-HGP44 antibody. P. gingivalis cells were grown in enriched BHI broth, washed with PBS, and suspended in PBS at an optical density at 540 nm of 0.8. Fifty-microliter suspensions were applied to each well of a microtiter plate and mixed with human erythrocyte suspensions and various concentrations of IgG.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 2. rHGP44B can agglutinate human erythrocytes. (A) Schematic diagram of HGP44 and truncated mutant proteins. (B) SDS-PAGE and CBB staining pattern of purified HGP44 and truncated mutant proteins expressed as His-tagged proteins. Lanes: M, molecular markers; A, HGP44A; B, HGP44B; C, HGP44C; D, HPG44D; E, HPG44E; F, HPG44F. (C) Purified rHGP44 and truncated mutant proteins were suspended with PBS at a concentration of 50 µg ml–1. The suspension and a series of twofold dilutions were applied to the wells of a microtiter plate and mixed with a human erythrocyte suspension and then incubated for 1 h at room temperature. Row A, HGP44A; row B, HGP44B; row C, HGP44C; row D, HGP44D; row E, HGP44E; row F, HGP44F.

Inhibition of hemagglutination by an interdomain regional peptide. Kelly et al. (24) mapped the hemagglutinating epitope of the HGP44 region at residues 1073 to 1112 since a synthetic peptide (GVRSPEAIRGRIQGTWRQKT) corresponding to residues 1083 to 1102 inhibited P. gingivalis cell-mediated hemagglutination efficiently and two peptides corresponding to residues 1073 to 1092 and residues 1093 to 1112 inhibited it less efficiently. We found in the present study that HGP44B without the hemagglutinating epitope induced hemagglutination, whereas HGP44A with the hemagglutinating epitope did not induce hemagglutination. Together, the results suggest that the interdomain amino acid region may not function as a hemagglutinating epitope but may function as a suppressor of hemagglutination. To examine this possibility, we investigated whether the peptide corresponding to residues 1083 to 1102 (Pep-1) inhibited HGP44B-mediated hemagglutination with human erythrocytes. We found that Pep-1 had inhibitory activity at concentrations of 0.2 to 12.5 mM, whereas Pep-1 with alanine substitutions (Pep-2) and Pep-3 corresponding to residues 745 to 764 did not (Fig. 3A and B). We also determined the interactions of the synthetic peptides with HGP44B by using an ELISA. The Pep-1 peptide bound HGP44B in a dose-dependent manner, whereas Pep-2 and Pep-3 did not (Fig. 3C). These results indicate that the interdomain amino acid region may interact with the HGP44B region to inhibit the hemagglutinating ability of HGP44B.


Figure 3
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 3. Effects of the interdomain regional peptide. (A) Amino acid sequences of the synthetic peptides Pep-1, Pep-2, and Pep-3. (B) Inhibitory effect of the interdomain regional peptide on HGP44B-mediated hemagglutination. HGP44B (10 µg) was mixed with various concentrations of the peptides and then incubated with 1% human erythrocytes for 1 h at room temperature. (C) Binding of the interdomain regional peptide to HGP44B. Microtiter plates were coated with various concentrations of the peptides for 2 h at 37°C. After blocking and washing, the plates were incubated with 0.1 µM HGP44B. Bound HGP44B was examined by an ELISA. The values are averages of three experiments. OD490, optical density at 490 nm.

HGP44B strongly agglutinates neuraminidase-treated erythrocytes. A previous study demonstrated that P. gingivalis cells strongly agglutinated neuraminidase-treated erythrocytes (36). We investigated whether neuraminidase treatment had a similar effect on HGP44B-mediated hemagglutination. Human erythrocytes were washed three times by centrifugation at 4°C in PBS and were treated with neuraminidase (10, 50, and 100 mU ml–1) in a 10% (vol/vol) cell suspension in PBS for 1 h at 37°C with gentle shaking. The neuraminidase-treated erythrocytes were washed three times with PBS. HGP44B-mediated hemagglutination was significantly enhanced by neuraminidase treatment in a dose-dependent manner (Fig. 4). The results suggested that removal of N-acetylneuraminic acid at the terminus of the carbohydrate structure enhanced the binding of HGP44B to erythrocytes.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. Effects of neuraminidase treatment of human erythrocytes on HGP44B-mediated hemagglutination. Neuraminidase (0, 10, 50, and 100 mU) was pretreated with 10% packed erythrocyte suspensions in PBS for 1 h at 37°C with gentle shaking. Erythrocytes were then washed three times by centrifugation with PBS. Then 100-µl portions of an HGP44B suspension and twofold serial dilutions of this suspension were applied to the wells of a microtiter plate and mixed with 100 µl of a 1% neuraminidase-treated erythrocyte suspension for 1 h at room temperature.

Determination of an HGP44B-targeted molecule on human erythrocytes. The membrane proteins prepared from normal erythrocytes produced multiple protein bands on SDS-PAGE gels as determined by CBB staining, as previously reported (Fig. 5A). The human erythrocyte membrane contains approximately 10 major classes of proteins, including spectrin, ankyrin, band 3, band 4.1, band 4.2, band 4.5, band 5, band 6, and band 7, which can be seen on CBB-stained gels (49, 50). The diffuse nature of band 3 and band 4.5 is due to heterogeneous biosynthetic glycosylation. Most of these proteins have been purified and characterized. Glycophorin, a highly sialylated glycoprotein, is a major constituent of human erythrocytes. Lutheran is a glycoprotein on the erythrocyte membrane that was recently characterized (12, 39). Glycophorin and lutheran protein bands are not detected by CBB staining. Of these proteins, band 3, band 4.5, band 7, glycophorin, and lutheran are intrinsic transmembrane proteins and are exposed to the external environment of the erythrocyte membrane. To determine what membrane protein of erythrocytes is a target of HGP44B, binding of HGP44B to these proteins was analyzed (Fig. 5). A solid-phase binding assay revealed that one major and two minor bands of erythrocyte membrane proteins reacted with HGP44B (Fig. 5B, middle panel). The mobility of the major band corresponded to that of a dimeric form of glycophorin AB (Fig. 5B, right panel).


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 5. HGP44B binds to an 89-kDa erythrocyte membrane protein. (A) SDS-PAGE patterns of human erythrocyte membrane ghosts. The gel was stained for protein with CBB. (B, left panel) Erythrocyte membrane polypeptides were transferred from polyacrylamide gradient gels to a PVDF membrane and were incubated with anti-band 3 antibody. (B, middle panel) Membrane polypeptides were transferred to a PVDF membrane and were incubated with HGP44B. Membrane proteins that interacted with HGP44 were detected with anti-HGP44 antibody. The arrow indicates the position of an 89-kDa membrane protein that interacted with HGP44. (B, right panel) Membrane polypeptides were transferred to a PVDF membrane and were incubated with anti-glycophorin A/B antibody. The glycophorin A2 homodimer (GPA2), glycophorin AB heterodimer (GPAB), and glycophorin B2 homodimer (GPB2) were detected.

Previous workers (28) have tried to determine the erythrocyte surface molecule that is a target for binding to P. gingivalis cells. Band 3 has been considered one of the targets, because treatment of erythrocytes with chymotrypsin resulted in disappearance of band 3 and a decrease in P. gingivalis-mediated hemagglutination (17). Inhibition of hemagglutinating activity by treatment of erythrocytes with chymotrypsin was also observed in the present study (Fig. 6A). Disappearance of band 3 from erythrocytes after the chymotrypsin treatment (Fig. 6B, left panel), as revealed by immunoblot analysis with anti-band 3, confirmed the findings of Hayashi et al. (17). However, we found that glycophorin was also degraded by the chymotrypsin treatment (Fig. 6B, right panel). The ability of HGP44B to agglutinate erythrocytes pretreated with purified RgpB proteinase was less than the ability of HGP44B to agglutinate intact erythrocytes (Fig. 6D), indicating that a target of HGP44B on erythrocytes might be degraded by the RgpB proteinase. Although CBB staining revealed no difference between erythrocyte membrane proteins treated with the RgpB proteinase and erythrocyte membrane proteins not treated with the RgpB proteinase (data not shown), immunoblot analysis with anti-glycophorin A/B and anti-band 3 showed that glycophorin A2 and glycophorin AB disappeared after treatment with the RgpB proteinase but that band 3 did not disappear (Fig. 6E). Glycophorins are identified as major bands stained with the Periodic acid-Schiff (PAS) reagent on SDS-PAGE gels (51). PAS staining also indicated that the levels of highly sialylated protein bands with high molecular masses were clearly decreased by the RgpB proteinase (Fig. 6F), suggesting that glycophorins A2 and AB were specifically degraded by RgpB.


Figure 6
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 6. Effects of treatment of erythrocytes with chymotrypsin or RgpB proteinase on HGP44B-mediated hemagglutination and profile of erythrocyte membrane proteins. (A) Hemagglutination. Human erythrocytes (50% [vol/vol] in PBS, 100 µl) were incubated with 1 mg ml–1 of chymotrypsin for 1 h at 37°C with gentle shaking. After the erythrocytes were washed with PBS for three times, they were mixed with HGP44B at concentrations of 0 to 10 µg per well for 1 h at room temperature. (B) Immunoblot analysis. Human erythrocytes (50% [vol/vol] in PBS, 100 µl) were incubated with chymotrypsin (0, 0.1, and 1.0 mg ml–1) for 1 h at 37°C with gentle shaking. The erythrocytes were then washed with PBS three times. Hemoglobin-free erythrocyte membrane white ghosts were then prepared from the washed erythrocytes and subjected to SDS-PAGE and immunoblot analysis with anti-band 3 antibody (left panel) and anti-glycophorin A/B antibody (right panel). GPA2, glycophorin A2 homodimer; GPAB, glycophorin AB heterodimer; GPB2, glycophorin B2 homodimer. (C) SDS-PAGE pattern of purified RgpB proteinase. The asterisk indicates the position of a protein band of RgpB. (D) Hemagglutination. Human erythrocytes (50% [vol/vol] in PBS, 100 µl) were incubated with purified RgpB (10 U) at 37°C overnight. An HGP44B suspension (10 µg per well) and twofold serial dilutions of this suspension were applied to the wells of a microtiter plate and mixed with an erythrocyte suspension for 1 h at room temperature. (E) Immunoblot analysis. White ghosts were prepared from the RgpB-treated erythrocytes and subjected to SDS-PAGE and immunoblot analysis with anti-band 3 and anti-glycophorin A/B antibodies. The left panel shows the results of an immunoblot analysis of normal membrane protein (left lane) and RgpB-treated membrane protein (right lane) with anti-band 3 antibody; the right panel shows the results of an immunoblot analysis of normal membrane protein (left lane) and RgpB-treated membrane protein (right lane) with anti-glycophorin A/B antibody. (F) PAS staining. Membrane ghosts were prepared from untreated erythrocytes (left lane) or RgpB-treated erythrocytes (right lane) and were subjected to SDS-PAGE and PAS staining.

We also investigated a target molecule on the erythrocyte membrane for HGP44B using a water-soluble, noncleavable, and membrane-impermeable cross-linker, BS3. HGP44B and erythrocyte surface proteins were cross-linked with BS3 as described in Materials and Methods. After cross-linking, erythrocytes were washed with PBS to remove the remaining HGP44B and BS3 and were then washed with 5 mM phosphate buffer (pH 8.0) to prepare membrane ghosts. Membrane ghosts were then disrupted with a lysis buffer. After HGP44 had been immunoprecipitated with rabbit anti-HGP44 antibody, HGP44 complexes were purified with protein A-Sepharose 4B and separated by SDS-PAGE, which was followed by an immunoblot analysis using mouse anti-HGP44 antibody (Fig. 7A), mouse anti-glycophorin A/B antibody (Fig. 7B), mouse anti-band 3 antibody (Fig. 7D), mouse anti-lutheran antibody (Fig. 7E), and mouse anti-glycophorin C antibody (Fig. 7F). Moreover, after the cross-linking experiment described above, glycophorin A/B complexes immunoprecipitated with mouse anti-glycophorin A/B antibody were analyzed with rabbit anti-HGP44 antibody (Fig. 7C). The immunoblots contained protein bands at high molecular masses (about 200 kDa) that appeared to be complexes of HGP44 and glycophorin (Fig. 7A, B, and C).


Figure 7
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 7. Cross-linking of HGP44B to erythrocyte membrane proteins. Packed human erythrocytes (500 µl) were incubated with HGP44B (200 µg) for 60 min at room temperature with gentle shaking. The chemical cross-linker BS3 was added and incubated for 30 min as described in Materials and Methods. Erythrocyte membrane ghosts were lysed and immunoprecipitated with rabbit anti-HGP44 antibody (A, B, D, E, and F) or mouse anti-glycophorin A/B (C). HGP44 was immunoprecipitated from erythrocyte membrane lysate, and the presence of HGP44 (A) and the association of glycophorin A/B (B), band 3 (D), lutheran (E), and glycophorin C (F) with HGP44 were revealed by Western blot analysis with corresponding antibodies. Glycophorin A/B was immunoprecipitated from erythrocyte membrane lysates, and the association of HGP44 with glycophorin A/B was revealed by Western blot analysis with anti-HGP44 (C). IP, immunoprecipitation; WB, Western blot analysis; GPC, glycophorin C; GP A/B, glycophorin A/B.

Next, we investigated whether exogenous glycophorin A or asialoglycophorin suppressed HGP44B-mediated hemagglutination of human erythrocytes. Glycophorin A and asialoglycophorin suppressed hemagglutination, whereas transferrin and albumin did not suppress hemagglutination. Asialoglycophorin suppressed hemagglutination more effectively than glycophorin A suppressed hemagglutination (Fig. 8). These results show that glycophorin A is the target molecule on erythrocytes for HGP44B. Interestingly, a nonerythroid sialylated protein, fetuin, and desialylated asialofetuin also suppressed hemagglutination, and asialofetuin suppressed it more effectively than fetuin suppressed it (Fig. 8).


Figure 8
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 8. Effects of various host proteins on HGP44B-mediated hemagglutination. HGP44B (2.5 µM) was mixed with various concentrations of glycophorin A, asialoglycophorin, fetuin, asialofetuin, transferrin, and albumin, and the mixtures were incubated with 1% human erythrocytes in PBS for 1 h at room temperature.

BIACORE analysis of the interaction between HGP44B and asialoglycophorin. To analyze the ability of HGP44B to bind glycophorin at the molecular level, the interaction between HGP44B and glycophorin was investigated using surface plasmon resonance detection. Glycophorin A is a major erythrocyte membrane sialoglycoprotein composed of 131 amino acids with 15 O-linked oligosaccharides attached to either serine or threonine at positions 1 to 50 and one N-linked oligosaccharide attached to the asparagine at position 26 (10, 43, 54, 55). Approximately 67% of sialic acids on human erythrocytes are borne on glycophorin A (6). Because of the nature of sialic acid-rich oligosaccharide, little glycophorin A was captured on the CM5 sensor chip used for surface plasmon resonance detection, so we could not perform the experiment examining the interaction of HGP44B with glycophorin A. To avoid the influence of sialic acids, we used asialoglycophorin instead of glycophorin A. Approximately 7,400 resonance units of asialoglycophorin were captured on the sensor chip after a 10-min injection of 100 µg ml–1 asialoglycophorin at a flow rate of 5 µl min–1. HGP44B at various concentrations was then injected for 3 min at a flow rate of 20 µl min–1. HGP44B was stably bound to asialoglycophorin in a dose-dependent manner (Fig. 9). The binding curves fit a 1:1 Langmuir binding model well. The dissociation constant was calculated to be 3.0 x 10–7 M. The results clearly indicate that HGP44B is able to bind asialoglycophorin.


Figure 9
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 9. Binding of HGP44B to asialoglycophorin immobilized on a CM5 sensor surface. The parameters of association and dissociation for HGP44B with asialoglycophorin were determined by surface plasmon resonance analysis (BIACORE). Sensorgrams for the binding of HGP44B to asialoglycophorin immobilized on a sensor chip were overlaid at various concentrations of HGP44B. HGP44B was injected at the concentrations (µg ml–1) indicated.


arrow
DISCUSSION
 
P. gingivalis-mediated hemagglutination has been studied by a number of researchers since it was discovered in 1974 (5, 20, 27, 29, 37, 38, 48); however, the precise mechanism of hemagglutination by P. gingivalis has not been completely elucidated. In this study, we obtained the first evidence that rHGP44 (HGP44B) itself has hemagglutinating activity. rgpA gene products are initially synthesized as a polyprotein with a high molecular mass and are further processed to small domains during translocation to the cell surface (45). Veith et al. (58) identified the processed domain proteins derived from rgpA using a matrix-assisted laser desorption ionization—time of flight mass spectrometer. Interestingly, a C-terminal portion of HGP44 that was originally described by Pavloff et al. (40) was not present in the processed HGP44 (RgpA44). The C-terminal residue of the processed HGP44 was predicted to be Ala1081, resulting from processing of Lys1082 by carboxypeptidase CPG70 (57). In the present study, we found that the fully processed HGP44 (HGP44B) had hemagglutinating activity but that the unprocessed HGP44 (HGP44A) did not have such activity and that a peptide derived from the interdomain region (Pep-1) between HGP44B and the following domain, HbR, could bind to HGP44B and inhibit its hemagglutinating activity. These results indicate that in the unprocessed RgpA polyprotein the interdomain regional residues might interact with HGP44 regional residues and inhibit hemagglutination until the molecule is translocated onto the cell surface, probably because the hemagglutinating activity affects normal cell functions in the periplasm. Previous studies have demonstrated that arginine and arginine-containing peptides, such as bradykinin, angiotensin I, and salmine sulfate, inhibit hemagglutinating activity (20, 33, 38). The HGP44A region has seven Arg residues. Four of these Arg residues are in the interdomain region (Fig. 1B). Pep-2, which has Ala residues in place of three Arg residues of Pep-1, was not able to inhibit HGP44B-mediated hemagglutination (Fig. 3B), suggesting that the cationic residues of the interdomain region are involved in inhibition of hemagglutination.

Shibata et al. (47) reported that the PVQNLT sequence in the HGP44B region is a motif for hemagglutination. O'Brien-Simpson et al. (34) have recently reported that the FEED and GTPNPNPNPNPNPNPGT sequences in the HGP44B region may have roles in binding to fibrinogen and collagen type V and in binding to hemoglobin, respectively. Both HGP44C and HGP44D have PVQNLT, FEED, and GTPNPNPNPNPNPNPGT sequences; however, they have no hemagglutinating activity. This suggests that a hemagglutinating epitope is located in the N-terminal sequence; however, we cannot rule out the possibility that there is a hemagglutinating epitope in the N-terminally truncated HGP44B derivatives, but the proteins cannot maintain functional configurations in solution because of the loss of the N-terminal sequence. Results obtained by using the cross-linker BS3 revealed that HGP44 interacted with glycophorin A/B (Fig. 7B and C) but not with band 3, lutheran, or glycophorin C (Fig. 7D, E, and F). Furthermore, exogenous glycophorin A inhibited HGP44B-mediated hemagglutination (Fig. 8). Degradation of glycophorin AB by chymotrypsin or RgpB reduced the ability of HGP44B to agglutinate erythrocytes (Fig. 6). These results suggest that glycophorin AB is a target molecule of HGP44B on the erythrocyte membrane. Hayashi et al. (17) previously reported that the band 3 protein isolated from erythrocyte membranes by the preparative PAGE method can inhibit P. gingivalis-induced hemagglutination and bind to P. gingivalis cells, indicating that band 3 is a receptor for P. gingivalis hemagglutinin. However, a dimeric form of glycophorin AB might have been present as a contaminant in their band 3 protein sample since glycophorin AB is not stained with CBB and since the molecular mass of a dimeric form of glycophorin AB (89 kDa) is very similar to that of band 3 (~90 kDa) (50).

Asialoglycophorin inhibited hemagglutination more effectively than glycophorin A inhibited hemagglutination, and asialofetuin inhibited hemagglutination more effectively than fetuin inhibited hemagglutination. These results are consistent with the results showing that neuraminidase-treated erythrocytes were easily agglutinated by HGP44B, suggesting that HGP44B tends to interact with desialylated glycoproteins. Glycophorin A contains approximately 15 O-linked sugar chains and one N-linked sugar chain (54). The structure of N-linked oligosaccharide consists of a dibranched complex-type sugar chain with N-acetylglucosamine linked at the mannosyl residue of the core portion and fucose linked at the proximal N-acetylglucosamine residue. More than 50% of sugar chains are monosialylated at one of two galactose terminal residues, and the other terminal galactose residue is not sialylated. The remaining N-linked sugar chains are disialylated (60). Therefore, HGP44 can agglutinate erythrocytes by binding to one unsialylated branch of an N-linked sugar chain in glycophorin A in the case of native erythrocytes and by binding to two desialylated branches of glycophorin A in the case of neuraminidase-treated erythrocytes. Fetuin contains six sugar chains, at least three O-linked sugar chains, and three N-linked sugar chains (7). The N-linked sugar chains are di- or tribranched complex-type oligosaccharides, and about 80% of these chains are sialylated (14). Like glycophorin A, HGP44 can bind to unsialylated sugar chains of fetuin. Transferrin contains two N-linked dibranched sugar chains that are fully sialylated (59). Albumin is not a glycoprotein. Therefore, our results suggest that HGP44 tends to bind unsialylated sugar chains.

Human erythrocytes have a definite life span of 120 days in circulation, after which they are captured and destroyed by macrophages. The sialic acids of membrane glycoconjugates play a key role in the life span of erythrocytes. Previous studies have shown that the content of surface-bound sialic acids depends on the age of erythrocytes (9, 15, 26). Young cells contain more sialic acid residues, while ageing in vivo reduces the number of these residues. Aminoff et al. (2, 18, 56) reported that glycopeptides rich in the disaccharide Gal-(ß1-3)GalNAc could be detected in old erythrocytes but not in young erythrocytes. They clearly demonstrated the role of glycophorin in ageing and sequestration of erythrocytes, as well as the sequential desialylation of glycophorins in the in vivo ageing of erythrocytes. Our results obtained using BIACORE indicated that HGP44 binds to asialoglycophorin, suggesting that HGP44 prefers to bind senescent erythrocytes.

Suzuki et al. (52) characterized the oligosaccharide cores of various erythrocytes using human anti-I serum and Ricinus communis agglutinin by fluorescence-activated cell sorting analysis. The human anti-I serum recognizes branched N-acetyllactosaminoglycans (blood group I-type antigens), and R. communis agglutinin specifically binds Gal-(ß1-4)GlcNAc- or Gal-(ß1-3)GalNAc-containing oligosaccharides (3). Fluorescence-activated cell sorting analysis of native and neuraminidase-treated erythrocytes indicated that erythrocytes of humans, cows, and guinea pigs contained sialyl-glycans with Gal-(ß1-4)GlcNAc or Gal-(ß1-3)GalNAc chains and branched N-acetyllactosaminoglycans with sialic acids but that equine erythrocytes contained no branched N-acetyllactosaminoglycans. Since native equine erythrocytes were not agglutinated by HGP44B (unpublished data), HGP44B may prefer branched N-acetyllactosaminoglycans. However, the hemagglutinating activity of HGP44B was not affected by the presence of exogenously added sugars, including D-galactose, D-glucose, N-acetyl-D-glucosamine, N-acetyl-D-lactosamine, galacto-N-biose, and lacto-N-biose, at a concentration of 100 mM (unpublished data). Filamentous hemagglutinin of Bordetella pertussis binds sulfated sugar (13). In Streptococcus gordonii, hemagglutinin binds an {alpha}-2,3-linked sialic acid-containing protein (53). In Tannerella forsythia (formerly Bacteroides forsythus), hemagglutination is completely inhibited by N-acetylneuraminidase at a concentration less than 10 mM (30). Helicobacter pylori hemagglutinin has a similar property (8). P. gingivalis HGP44 hemagglutinin described here seems to differ from the hemagglutinins of other bacterial species. Further investigations are needed to determine the structure of the binding site of erythrocytes for P. gingivalis HGP44 hemagglutinin.

In addition to the ability to agglutinate erythrocytes, HGP44B can bind to host proteins, such as fibronectin, fibrinogen, laminin, and collagen type V (unpublished data). These matrix proteins have been reported to coat epithelial, endothelial, and fibroblast cells. Binding of HGP44B to these matrix proteins may be important for invasion of host cells and tissues by P. gingivalis. We show here that the interdomain regional peptide directly binds HGP44B and inhibits its hemagglutinating activity. The peptide also suppresses the ability of HGP44B to bind host proteins mentioned above (unpublished data). These results suggest that the interdomain regional peptide prevents adherence of P. gingivalis cells to host cells and tissues, which is the initial step in P. gingivalis infection.


arrow
ACKNOWLEDGMENTS
 
We thank Mihoko Nonaka, Nagasaki University Graduate School of Biomedical Science, and Mikiko Matsuda, Dental Hospital of Nagasaki University, for technical assistance. We also thank Naoya Ohara, Mikio Shoji, and Hideharu Yukitake, Division of Microbiology and Oral Infection, for helpful suggestions.

This work was supported by grant-in-aid 17209057 for scientific research from the Ministry of Education, Science, Sports and Culture, Japan.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Sakamoto 1-7-1, Nagasaki 852-8588, Japan. Phone: 81-95-849-7649. Fax: 81-95-849-7650. E-mail: knak{at}nagasaki-u.ac.jp Back

{triangledown} Published ahead of print on 23 March 2007. Back


arrow
REFERENCES
 
    1
  1. Aduse-Opoku, J., J. M. Slaney, M. Rangarajan, J. Muir, K. A. Young, and M. A. Curtis. 1997. The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin. J. Bacteriol. 179:4778-4788.[Abstract/Free Full Text]
    2. 2
  2. Aminoff, D. 1988. The role of sialoglycoconjugates in the aging and sequestration of red cells from circulation. Blood Cells 14:229-257.[Medline]
    3. 3
  3. Baenziger, J. U., and D. Fiete. 1979. Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. J. Biol. Chem. 254:9795-9799.[Free Full Text]
    4. 4
  4. Booth, V., and T. Lehner. 1997. Characterization of the Porphyromonas gingivalis antigen recognized by a monoclonal antibody which prevents colonization by the organism. J. Periodontal Res. 32:54-60.[Medline]
    5. 5
  5. Boyd, J., and B. C. McBride. 1984. Fractionation of hemagglutinating and bacterial binding adhesins of Bacteroides gingivalis. Infect. Immun. 45:403-409.[Abstract/Free Full Text]
    6. 6
  6. Bretscher, M. S. 1971. A major protein which spans the human erythrocyte membrane. J. Mol. Biol. 59:351-357.[CrossRef][Medline]
    7. 7
  7. Carr, S. A., M. J. Huddleston, and M. F. Bean. 1993. Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry. Protein Sci. 2:183-196.[Medline]
    8. 8
  8. Chaturvedi, G., R. Tewari, Mrigank, N. Agnihotri, R. A. Vishwakarma, and N. K. Ganguly. 2001. Inhibition of Helicobacter pylori adherence by a peptide derived from neuraminyl lactose binding adhesin. Mol. Cell. Biochem. 228:83-89.[CrossRef][Medline]
    9. 9
  9. Cohen, N. S., J. E. Ekholm, M. G. Luthra, and D. J. Hanahan. 1976. Biochemical characterization of density-separated human erythrocytes. Biochim. Biophys. Acta 419:229-242.[Medline]
    10. 10
  10. Dahr, W., G. Uhlenbruck, E. Janssen, and R. Schmalisch. 1976. Heterogeneity of human cell membrane sialoglycoproteins. Blut 32:171-184.[CrossRef][Medline]
    11. 11
  11. Fairbanks, G., T. L. Steck, and D. F. Wallach. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606-2617.[CrossRef][Medline]
    12. 12
  12. Gauthier, E., C. Rahuel, M. P. Wautier, W. El Nemer, P. Gane, J. L. Wautier, J. P. Cartron, Y. Colin, and C. Le Van Kim. 2005. Protein kinase A-dependent phosphorylation of Lutheran/basal cell adhesion molecule glycoprotein regulates cell adhesion to laminin alpha5. J. Biol. Chem. 280:30055-30062.[Abstract/Free Full Text]
    13. 13
  13. Geuijen, C. A., R. J. Willems, and F. R. Mooi. 1996. The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars. Infect. Immun. 64:2657-2665.[Abstract/Free Full Text]
    14. 14
  14. Green, E. D., G. Adelt, J. U. Baenziger, S. Wilson, and H. Van Halbeek. 1988. The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. J. Biol. Chem. 263:18253-18268.[Abstract/Free Full Text]
    15. 15
  15. Gutowski, K. A., J. L. Hudson, and D. Aminoff. 1991. Flow cytometric analysis of human erythrocytes. I. Probed with lectins and immunoglobulins. Exp. Gerontol. 26:315-326.[CrossRef][Medline]
    16. 16
  16. Han, N., J. Whitlock, and A. Progulske-Fox. 1996. The hemagglutinin gene A (hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, direct repeats. Infect. Immun. 64:4000-4007.[Abstract/Free Full Text]
    17. 17
  17. Hayashi, H., A. Nagata, D. Hinode, M. Sato, and R. Nakamura. 1992. Survey of a receptor protein in human erythrocytes for hemagglutinin of Porphyromonas gingivalis. Oral Microbiol. Immunol. 7:204-211.[Medline]
    18. 18
  18. Henrich, C. J., and D. Aminoff. 1983. Isolation and characterization of a glycopeptide from human senescent erythrocytes. Carbohydr Res. 120:55-66.[CrossRef][Medline]
    19. 19
  19. Holt, S. C., and T. E. Bramanti. 1991. Factors in virulence expression and their role in periodontal disease pathogenesis. Crit. Rev. Oral Biol. Med. 2:177-281.[Abstract/Free Full Text]
    20. 20
  20. Inoshita, E., A. Amano, T. Hanioka, H. Tamagawa, S. Shizukuishi, and A. Tsunemitsu. 1986. Isolation and some properties of exohemagglutinin from the culture medium of Bacteroides gingivalis 381. Infect. Immun. 52:421-427.[Abstract/Free Full Text]
    21. 21
  21. Isogai, H., E. Isogai, F. Yoshimura, T. Suzuki, W. Kagota, and K. Takano. 1988. Specific inhibition of adherence of an oral strain of Bacteroides gingivalis 381 to epithelial cells by monoclonal antibodies against the bacterial fimbriae. Arch. Oral Biol. 33:479-485.[CrossRef][Medline]
    22. 22
  22. Kadowaki, T., M. Yoneda, K. Okamoto, K. Maeda, and K. Yamamoto. 1994. Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphyromonas gingivalis. J. Biol. Chem. 269:21371-21378.[Abstract/Free Full Text]
    23. 23
  23. Kamaguchi, A., T. Ohyama, E. Sakai, R. Nakamura, T. Watanabe, H. Baba, and K. Nakayama. 2003. Adhesins encoded by the gingipain genes of Porphyromonas gingivalis are responsible for co-aggregation with Prevotella intermedia. Microbiology 149:1257-1264.[Abstract/Free Full Text]
    24. 24
  24. Kelly, C. G., V. Booth, H. Kendal, J. M. Slaney, M. A. Curtis, and T. Lehner. 1997. The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis. Clin. Exp. Immunol. 110:285-291.[Medline]
    25. 25
  25. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
    26. 26
  26. Luner, S. J., D. Szklarek, R. J. Knox, G. V. Seaman, J. Y. Josefowicz, and B. R. Ware. 1977. Red cell charge is not a function of cell age. Nature 269:719-721.[CrossRef][Medline]
    27. 27
  27. Mayrand, D., B. C. McBride, T. Edwards, and S. Jensen. 1980. Characterization of Bacteroides asaccharolyticus and B. melaninogenicus oral isolates. Can. J. Microbiol. 26:1178-1183.[Medline]
    28. 28
  28. Mouton, C., and F. Chandad. 1993. Hemagglutination and hemagglutinins, p. 199-217. In H. N. Shah, D. Mayrand, and R. J. Genco (ed.), Biology of the species Porphyromonas gingivalis. CRC Press, Boca Raton, FL.
    29. 29
  29. Mouton, C., D. Ni Eidhin, M. Deslauriers, and L. Lamy. 1991. The hemagglutinating adhesin HA-Ag2 of Bacteroides gingivalis is distinct from fimbrilin. Oral Microbiol. Immunol. 6:6-11.[Medline]
    30. 30
  30. Murakami, Y., N. Higuchi, H. Nakamura, F. Yoshimura, and F. G. Oppenheim. 2002. Bacteroides forsythus hemagglutinin is inhibited by N-acetylneuraminyllactose. Oral Microbiol. Immunol. 17:125-128.[CrossRef][Medline]
    31. 31
  31. Nakayama, K., T. Kadowaki, K. Okamoto, and K. Yamamoto. 1995. Construction and characterization of arginine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis. Evidence for significant contribution of Arg-gingipain to virulence. J. Biol. Chem. 270:23619-23626.[Abstract/Free Full Text]
    32. 32
  32. Nakayama, K., D. B. Ratnayake, T. Tsukuba, T. Kadowaki, K. Yamamoto, and S. Fujimura. 1998. Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis. Mol. Microbiol. 27:51-61.[CrossRef][Medline]
    33. 33
  33. Nishikata, M., and F. Yoshimura. 1991. Characterization of Porphyromonas (Bacteroides) gingivalis hemagglutinin as a protease. Biochem. Biophys. Res. Commun. 178:336-342.[CrossRef][Medline]
    34. 34
  34. O'Brien-Simpson, N. M., R. D. Pathirana, R. A. Paolini, Y. Y. Chen, P. D. Veith, V. Tam, N. Ally, R. N. Pike, and E. C. Reynolds. 2005. An immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced periodontal bone loss. J. Immunol. 175:3980-3989.[Abstract/Free Full Text]
    35. 35
  35. Ogawa, T., and S. Hamada. 1994. Hemagglutinating and chemotactic properties of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis. Infect. Immun. 62:3305-3310.[Abstract/Free Full Text]
    36. 36
  36. Okuda, K., M. Ono, and T. Kato. 1989. Neuraminidase-enhanced attachment of Bacteroides intermedius to human erythrocytes and buccal epithelial cells. Infect. Immun. 57:1635-1637.[Abstract/Free Full Text]
    37. 37
  37. Okuda, K., and I. Takazoe. 1974. Haemagglutinating activity of Bacteroides melaninogenicus. Arch. Oral Biol. 19:415-416.[CrossRef][Medline]
    38. 38
  38. Okuda, K., A. Yamamoto, Y. Naito, I. Takazoe, J. Slots, and R. J. Genco. 1986. Purification and properties of hemagglutinin from culture supernatant of Bacteroides gingivalis. Infect. Immun. 54:659-665.[Abstract/Free Full Text]
    39. 39
  39. Parsons, S. F., G. Mallinson, C. H. Holmes, J. M. Houlihan, K. L. Simpson, W. J. Mawby, N. K. Spurr, D. Warne, A. N. Barclay, and D. J. Anstee. 1995. The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver. Proc. Natl. Acad. Sci. USA 92:5496-5500.[Abstract/Free Full Text]
    40. 40
  40. Pavloff, N., J. Potempa, R. N. Pike, V. Prochazka, M. C. Kiefer, J. Travis, and P. J. Barr. 1995. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonas gingivalis. Biosynthesis as a proteinase-adhesin polyprotein. J. Biol. Chem. 270:1007-1010.[Abstract/Free Full Text]
    41. 41
  41. Pike, R., W. McGraw, J. Potempa, and J. Travis. 1994. Lysine- and arginine-specific proteinases from Porphyromonas gingivalis. Isolation, characterization, and evidence for the existence of complexes with hemagglutinins. J. Biol. Chem. 269:406-411.[Abstract/Free Full Text]
    42. 42
  42. Pike, R. N., J. Potempa, W. McGraw, T. H. Coetzer, and J. Travis. 1996. Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis. J. Bacteriol. 178:2876-2882.[Abstract/Free Full Text]
    43. 43
  43. Pisano, A., J. W. Redmond, K. L. Williams, and A. A. Gooley. 1993. Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A. Glycobiology 3:429-435.[Abstract/Free Full Text]
    44. 44
  44. Potempa, J., R. Pike, and J. Travis. 1995. The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain. Infect. Immun. 63:1176-1182.[Abstract/Free Full Text]
    45. 45
  45. Sato, K., E. Sakai, P. D. Veith, M. Shoji, Y. Kikuchi, H. Yukitake, N. Ohara, M. Naito, K. Okamoto, E. C. Reynolds, and K. Nakayama. 2005. Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis. J. Biol. Chem. 280:8668-8677.[Abstract/Free Full Text]
    46. 46
  46. Shi, Y., D. B. Ratnayake, K. Okamoto, N. Abe, K. Yamamoto, and K. Nakayama. 1999. Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA. J. Biol. Chem. 274:17955-17960.[Abstract/Free Full Text]
    47. 47
  47. Shibata, Y., M. Hayakawa, H. Takiguchi, T. Shiroza, and Y. Abiko. 1999. Determination and characterization of the hemagglutinin-associated short motifs found in Porphyromonas gingivalis multiple gene products. J. Biol. Chem. 274:5012-5020.[Abstract/Free Full Text]
    48. 48
  48. Slots, J., and R. J. Gibbons. 1978. Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. Infect. Immun. 19:254-264.[Abstract/Free Full Text]
    49. 49
  49. Steck, T. L. 1978. The band 3 protein of the human red cell membrane: a review. J. Supramol Struct. 8:311-324.[CrossRef][Medline]
    50. 50
  50. Steck, T. L. 1974. The organization of proteins in the human red blood cell membrane. A review. J. Cell Biol. 62:1-19.[Free Full Text]
    51. 51
  51. Steck, T. L., and G. Dawson. 1974. Topographical distribution of complex carbohydrates in the erythrocyte membrane. J. Biol. Chem. 249:2135-2142.[Abstract/Free Full Text]
    52. 52
  52. Suzuki, T., A. Portner, R. A. Scroggs, M. Uchikawa, N. Koyama, K. Matsuo, Y. Suzuki, and T. Takimoto. 2001. Receptor specificities of human respiroviruses. J. Virol. 75:4604-4613.[Abstract/Free Full Text]
    53. 53
  53. Takahashi, Y., A. L. Sandberg, S. Ruhl, J. Muller, and J. O. Cisar. 1997. A specific cell surface antigen of Streptococcus gordonii is associated with bacterial hemagglutination and adhesion to alpha2-3-linked sialic acid-containing receptors. Infect. Immun. 65:5042-5051.[Abstract/Free Full Text]
    54. 54
  54. Tomita, M., H. Furthmayr, and V. T. Marchesi. 1978. Primary structure of human erythrocyte glycophorin A. Isolation and characterization of peptides and complete amino acid sequence. Biochemistry 17:4756-4770.[CrossRef][Medline]
    55. 55
  55. Tomita, M., and V. T. Marchesi. 1975. Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc. Natl. Acad. Sci. USA 72:2964-2968.[Abstract/Free Full Text]
    56. 56
  56. Vaysse, J., L. Gattegno, D. Bladier, and D. Aminoff. 1986. Adhesion and erythrophagocytosis of human senescent erythrocytes by autologous monocytes and their inhibition by beta-galactosyl derivatives. Proc. Natl. Acad. Sci. USA 83:1339-1343.[Abstract/Free Full Text]
    57. 57
  57. Veith, P. D., Y. Y. Chen, and E. C. Reynolds. 2004. Porphyromonas gingivalis RgpA and Kgp proteinases and adhesins are C terminally processed by the carboxypeptidase CPG70. Infect. Immun. 72:3655-3657.[Abstract/Free Full Text]
    58. 58
  58. Veith, P. D., G. H. Talbo, N. Slakeski, S. G. Dashper, C. Moore, R. A. Paolini, and E. C. Reynolds. 2002. Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50. Biochem. J. 363:105-115.[CrossRef][Medline]
    59. 59
  59. Yamashita, K., H. Ideo, T. Ohkura, K. Fukushima, I. Yuasa, K. Ohno, and K. Takeshita. 1993. Sugar chains of serum transferrin from patients with carbohydrate deficient glycoprotein syndrome. Evidence of asparagine-N-linked oligosaccharide transfer deficiency. J. Biol. Chem. 268:5783-5789.[Abstract/Free Full Text]
    60. 60
  60. Yoshima, H., H. Furthmayr, and A. Kobata. 1980. Structures of the asparagine-linked sugar chains of glycophorin A. J. Biol. Chem. 255:9713-9718.[Abstract/Free Full Text]
    61. 61
  61. Yoshimura, F., K. Takahashi, Y. Nodasaka, and T. Suzuki. 1984. Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis. J. Bacteriol. 160:949-957.[Abstract/Free Full Text]

Journal of Bacteriology, June 2007, p. 3977-3986, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01691-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sato, K., Kido, N., Murakami, Y., Hoover, C. I., Nakayama, K., Yoshimura, F. (2009). Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis. Microbiology 155: 1282-1293 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakai, E.
Right arrow Articles by Nakayama, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakai, E.
Right arrow Articles by Nakayama, K.

Copyright © 2010 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»