Active domains of salivary statherin on apatitic surfaces for binding to Fusobacterium nucleatum cells

Shinichi Sekine1, Kosuke Kataoka1, Muneo Tanaka1, Hideki Nagata1, Toru Kawakami2, Kenichi Akaji2, Saburo Aimoto2 and Satoshi Shizukuishi1

1 Departments of Preventive Dentistry, Graduate School of Dentistry, Osaka University, Suita, Japan
2 Institute for Protein Research, Osaka University, Suita, Japan

Correspondence
Satoshi Shizukuishi
shizuku{at}dent.osaka-u.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fusobacterium nucleatum can bind to saliva-coated tooth surfaces. However, the nature of the domains of salivary protein that interact with F. nucleatum remains unclear. The ability of individual proteins in human submandibular-sublingual saliva (HSMSL) to bind F. nucleatum cells was examined by dot blot assay; statherin displayed the strongest binding activity. Statherin binding sites were determined based on binding of 125I-labelled F. nucleatum to statherin-coated hydroxyapatite (sHAP) beads via inhibition assays using synthetic analogous peptide fragments of whole statherin. Analogous peptides corresponding to residues 19–26 and 32–39 of statherin inhibited binding by 77 % and 68 %, respectively. Synthetic peptides were also prepared by serial deletions of individual residues from N- and C-termini of the peptides GPYQPVPE (aa 19–26) and QPYQPQYQ (aa 32–39). The inhibitory effects of peptides YQPVPE (aa 21–26) and PYQPQYQ (aa 33–39) were very similar to those of GPYQPVPE and QPYQPQYQ, respectively. However, additional deletion of residues resulted in significant reduction of the inhibitory effect. Alanine-scan analysis of YQPVPE revealed that all tested peptides retained inhibitory activity; only YAPVPE exhibited significantly decreased inhibitory activity. These findings suggest that YQPVPE and PYQPQYQ may represent the minimal active segments of statherin for binding to F. nucleatum; moreover, Gln may be a key amino acid in the active segment.


Abbreviations: HAP, hydroxyapatite; HSMSL, human submandibular-sublingual saliva; PRGP, proline-rich glycoprotein; PRP1, acidic proline-rich protein-1; sHAP, statherin-coated hydroxyapatite


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fusobacterium nucleatum, which plays an important role in early colonization during plaque formation, acts as a bridge between salivary proteins and other co-aggregating strains, or between early colonizers and late colonizers (Kolenbrander & London, 1993; Bradshaw et al., 1998). F. nucleatum is believed to be a predisposing factor for the initiation of periodontal disease.

Saliva coats the surfaces of components lining the oral cavity such as mucosal membrane, healthy crevices and supra- and subgingival plaques; moreover, saliva appears to be critical to micro-organisms in terms of adhesion to and colonization of the oral cavity (Gibbons & Hay, 1988; Scannapieco, 1994). Among salivary proteins, statherin is a unique acidic, carbohydrate-free phosphoprotein (Hay & Moreno, 1989). It inhibits primary and secondary precipitation of calcium salts; additionally, statherin is tightly adsorbed to enamel surfaces (Johnsson et al., 1993). Statherin promotes bacterial adhesion by organisms including Actinomyces viscosus (Gibbons et al., 1990; Niemi & Johansson, 2004), Actinomyces naeslundii (Strömberg et al., 1992; Niemi & Johansson, 2004) and Porphyromonas gingivalis (Amano et al., 1994) to surfaces such as hydroxyapatite (HAP) when it is pre-adsorbed to these surfaces.

Xie et al. (1991) demonstrated enhanced adsorption of F. nucleatum to statherin-coated hydroxyapatite (sHAP); however, the nature of statherin domain interaction with F. nucleatum remains unclear. The aim of the present investigation was to examine the ability of human submandibular-sublingual saliva (HSMSL) components, including statherin, to bind to F. nucleatum cells, and to delineate the active binding segments of the statherin molecule (bound to HAP) involved in interactions with F. nucleatum cells.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial culture conditions and radiolabelling.
F. nucleatum ATCC 10953 was grown in trypticase soy broth supplemented with yeast extract (1 mg ml–1), haemin (5 µg ml–1) and menadione (1 µg ml–1) at 37 °C under anaerobic conditions (80 % N2, 10 % CO2, 10 % H2). Following harvest, bacterial cells were washed three times with 50 mM KCl solution containing 1 mM KH2PO4, 1 mM CaCl2 and 0·1 mM MgCl2 (pH 6·8 KCl buffer). Cells were radiolabelled with Na125I using the Lactoperoxidase 125I-labelling kit (ICN Pharmaceuticals; Lee et al., 1992). The specific activity of the iodinated protein was 1·6 mCi (59·2 MBq) per 109 cells.

Purification of salivary components and preparation of synthetic statherin peptides.
Statherin, acidic proline-rich protein-1 (PRP1) and proline-rich glycoprotein (PRGP) were purified from HSMSL collected from three male donors as described previously (Ramasubbu et al., 1991). Ten analogous peptide fragments (peptides 1–14, 14–26, 26–43, 1–6, 6–14, 14–21, 19–26, 26–34, 32–39 and 38–43) corresponding to the amino acid sequence of statherin were synthesized commercially (Table 1, Kataoka et al., 1997). Peptides 1–14 (1–6 and 6–14) corresponded to the N-terminal helix domain; peptides 14–26 (14–21 and 19–26) were contained in the middle region of statherin; and peptides 26–43 (26–34, 32–39 and 38–43) were used as C-terminal-derived peptides. Twenty-six analogous peptides, including the deletion and alanine-scan peptides, were synthesized and purified in succession (Aimoto, 1989). The amino acid composition and mass of products were confirmed with an L-8500 amino acid analyser (Hitachi) and by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry employing the Voyager-DE Bio-spectrometry Workstation of Perceptive Biosystems (PerkinElmer Biosystems).


View this table:
[in this window]
[in a new window]
 
Table 1. Amino acid sequence of statherin and its synthetic peptides

 
Identification of salivary proteins binding to F. nucleatum cells.
HSMSL was dissolved in SDS sample buffer, with heating, followed by separation by SDS-PAGE on a 15–25 % polyacrylamide gel and staining with Coomassie Brilliant Blue. To identify salivary proteins binding to F. nucleatum cells, HSMSL separated by SDS-PAGE was immobilized on a nitrocelullose membrane with a semi-dry transfer system (Semi-phore TE-77, Hoefer Scientific Instruments) according to the manufacturer's instructions. Unoccupied binding sites on the membranes were blocked by incubation for 1 h with Tris-buffered saline (20 mM Tris/HCl, 0·154 M NaCl, pH 7·5) containing 10 % Block Ace (casein solution prepared from homogenized milk, Snow Brand), which served as a blocking agent. Membranes were incubated with 5 ml of 125I-labelled F. nucleatum (125I-F. nucleatum; 5x108 cells ml–1) overnight at 4 °C. Binding of 125I-F. nucleatum to salivary components was detected with X-ray film as described previously (Amano et al., 1994).

Dot blot assay.
Binding ability to salivary proteins was determined with dot blot assay involving activated Immobilon-P (PVDF membrane, Bio-Rad) as described previously (Kataoka et al., 1997). Each salivary protein (PRGP, PRP1, statherin) or 2 % lipid-free bovine serum albumin (BSA A-7030, Sigma), which was dissolved in KCl buffer, was loaded into wells of a Bio-Dot apparatus (Bio-Rad) by gentle aspiration. The amounts of salivary proteins immobilized on the membrane were optimized so as to be equivalent by amino acid analysis in a manner consistent with the approach of Amano et al. (1996a). Membranes were blocked with 5 % Block Ace and incubated with 3 ml of 125I-F. nucleatum (5x108 cells ml–1) overnight at room temperature. Subsequently, membranes were washed with KCl buffer containing 100 mM NaCl and subjected to autoradiography according to the method of Amano et al. (1996b). Density was estimated with the NIH Image program (National Technical Information Service) as described by Kuboniwa et al. (1998).

Inhibition of F. nucleatum binding to statherin by various compounds and peptides.
Assay of 125I-F. nucleatum cell binding to sHAP beads was conducted according to the method described previously by Kataoka et al. (1999). Briefly, 3 mg HAP beads in a tube were incubated with 100 µl statherin solution (100 µg ml–1) overnight at room temperature. An inhibitor [either L-arginine (100 µl, 0·1 M), histidine (100 µl, 0·1 M), sodium chloride (100 µl, 1 mM; control), peptide solution (100 µl, 1 mM) or statherin (100 µl, 1 mM)] and 100 µl of 125I-F. nucleatum (5x108 cells ml–1) were introduced into the tube containing sHAP beads and incubated for 1 h at room temperature. The specific binding level was calculated by subtracting the nonspecific binding level, which was obtained by pre-incubation of sHAP beads with unlabelled whole cells (200 µl of 5x108 F. nucleatum cells ml–1) for 1 h at room temperature, from the binding level corresponding to labelled whole cells. The inhibitory rate was calculated by comparison of the specific binding levels with and without inhibitors. All assays were performed in triplicate on three separate occasions.

Statistical methods.
The data were averaged (means±standard deviation). Comparisons were performed using Student's t test and P values of <0·01 were considered significant.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of salivary components binding to F. nucleatum
A solid-phase assay was conducted to identify salivary proteins binding to F. nucleatum cells in HSMSL. Three components of HSMSL, statherin, PRP1 and PRGP, exhibited significant binding to F. nucleatum cells (Fig. 1a). A nitrocellulose membrane adsorbed with an identical amount of these three purified proteins and BSA was incubated with 125I-F. nucleatum to confirm binding ability. Binding activity was visualized by autoradiography (Fig. 1b) and estimated by relative densitometric analysis of reaction dots with the NIH Image program. When the density of a dot on the membrane was adjusted to 100 % for statherin and 0 % for BSA, density was 82 % and 47 % for PRGP and for PRP1, respectively. These results suggest that, among the salivary proteins, statherin (on solid surfaces) may bind most strongly to F. nucleatum.



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 1. Binding of HSMSL components to F. nucleatum cells. (a) SDS-PAGE profile. Lane 1, molecular mass standard (std); lane 2, HSMSL protein separated by SDS-PAGE stained with Coomassie Brilliant Blue; lane 3, autoradiogram of the salivary components bound to 125I-F. nucleatum (5x108 cells ml–1) on a nitrocellulose membrane. (b) Dot blot assays for determination of binding activity of the purified components, i.e. PRGP, PRP1, statherin and BSA (as a negative control) to 125I-F. nucleatum on a PVDF membrane.

 
Inhibition of the binding of F. nucleatum cells to sHAP by various compounds
To assess the nature of the interactions between statherin and 125I-F. nucleatum, the effects of charged amino acids [L-arginine (0·1 M) and histidine (0·1 M) and sodium chloride (1 mM) were examined via addition to reaction mixtures. Although L-arginine inhibited binding by 14±0·9 %, the inhibitory effects of histidine and NaCl were 4±0·4 % and 3±0·2 %, respectively.

Determination of the binding regions of statherin utilizing analogous peptides
Three synthetic peptides corresponding to amino acid residues 1–14, 14–26 and 26–43 of the statherin molecule (Table 1) were employed to identify the segment of statherin involved in binding to F. nucleatum (Fig. 2). The inhibitory effects of these three fragments and whole statherin were examined in the binding of 125I-F. nucleatum cells to sHAP. The inhibitory effect of whole statherin was negligible, suggesting that the binding region of statherin might be cryptic. Peptide 14–26 displayed significant inhibition of 48 %; peptide 26–43 inhibited whole-cell binding by 38 %, whereas the inhibitory effect of peptide 1–14 was 26 %.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. Inhibitory effect of synthetic statherin fragments on the binding of 125I-F. nucleatum to sHAP. sHAP beads were incubated with 200 µl of 125I-F. nucleatum cells (5x108 cells ml–1) in the presence of statherin (100 µl, I mM) or of each fragment (100 µl, I mM). All assays were performed in triplicate on three separate occasions. Data are expressed as means±standard deviations. *Significantly different from level of control (without peptides or statherin), P<0·01.

 
To establish further localization of the active binding regions, seven synthetic analogous peptides corresponding to residues 1–6, 6–14, 14–21, 19–26, 26–34, 32–39 and 38–43 of statherin (Table 1) were prepared. The inhibitory effects of these seven peptides were also examined in the binding of 125I-F. nucleatum cells to sHAP (Fig. 2). Peptides 19–26 and 32–39 demonstrated significant inhibition of binding, 77 % and 68 %, respectively.

Determination of the minimal active segments in residues 19–26 (GPYQPVPE) and 32–39 (QPYQPQYQ)
Serial peptides, in which amino acid residues were deleted from the N-terminus of peptide 19–26 and from the C-terminus of peptide 21–26, were synthesized to identify the minimal active segment of peptide 19–26 (Fig. 3). Serial deletions of the Gly and Pro residues in peptide 19–26 (GPYQPVPE) displayed no effect. Deletion of the Tyr residue, however, led to dramatic reduction of the inhibitory effect of the peptide. Furthermore, examination of the significance of the C-terminal residues revealed complete loss of activity following deletion of the Glu residue in peptide 21–26 (YQPVPE).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Determination of the active binding domain in GPYQPVPE (aa 19–26) of statherin. The experimental procedure was as described in the legend of Fig. 2. Synthetic peptides, in which amino acid residues were sequentially deleted from the N-terminus of peptide GPYQPVPE and the C-terminus of peptide YQPVPE, were investigated as inhibitors to the binding of 125I-F. nucleatum to sHAP beads. All assays were performed in triplicate on three separate occasions. Data are expressed as means±standard deviations. *Significantly different from level of control (without peptides or statherin), P<0·01.

 
Additionally, the contribution of specific amino acids to binding was determined by systematic replacement of each amino acid in the YQPVPE sequence with Ala. All alanine-scan peptides of YQPVPE retained inhibitory activity; only YAPVPE exhibited significantly decreased inhibitory activity in comparison with YQPVPE (Fig. 4). This result suggests that Gln may be a key amino acid residue in terms of segment activity. As shown in Fig. 5, serial deletion of the Gln and the Pro from the N-terminus in peptide 32–39 (QPYQPQYQ) and deletion of the Gln from the C-terminus in peptide 33–39 (PYQPQYQ) led to loss of the inhibitory effect. These findings strongly suggest that YQPVPE and PYQPQYQ are the minimal active segments for binding to 125I-F. nucleatum cells.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Alanine-scan technique was employed for identification of amino acid residues of YQPVPE (aa 21–26) involved in inhibition. Alanine-scan peptides served as inhibitors in the binding of 125I-F. nucleatum to sHAP beads. The experimental procedure was as described in the legend of Fig. 2. All assays were performed in triplicate on three separate occasions. Data are expressed as means±standard deviations. a vs b, P<0·0001; a vs c, P<0·001; d vs e, P<0·01.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Determination of the active binding domain in residues QPYQPQYQ (aa 32–39) of statherin. The experimental procedure was as described in the legend of Fig. 2. Synthetic peptides, in which amino acid residues were sequentially deleted from the N-terminus of peptide QPYQPQYQ and the C-terminus of peptide PYQPQYQ, were investigated as inhibitors of the binding of 125I-F. nucleatum to sHAP beads. All assays were performed in triplicate on three separate occasions. Data are expressed as means±standard deviations. *Significantly different from level of control (without peptides or statherin), P<0·01.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The present investigation established that three components of HSMSL, statherin, PRP1 and PRGP, bind to F. nucleatum cells (Fig. 1a). In particular, statherin, on a solid surface, displayed the strongest binding ability to F. nucleatum among the salivary proteins (Fig. 1b). The presence of statherin in acquired pellicle in vivo (Yao et al., 2001, 2003) and the concentration (mean 12·8 µM, range 3·0–27·3) of statherin in human saliva (Hay & Moreno, 1989) indicate that binding between F. nucleatum and statherin is significant with respect to dental plaque formation.

Strains of F. nucleatum bind to epithelial cells (Murray et al., 1988), to gingival fibroblasts (Falkler et al., 1982) and to the surface of certain other bacteria (Falkler et al., 1981; Kaufman & DiRienzo, 1988). Several researchers have explored a primarily lectin-like interaction on F. nucleatum in terms of F. nucleatum binding mechanisms (Murray et al., 1988; Kolenbrander & Andersen, 1989; Han et al., 2000). However, as statherin contains no carbohydrate residues, the interaction between F. nucleatum and statherin may not be similar to the lectin-like interaction. On the other hand, Dehazya & Coles (1980) reported that the haemagglutination properties of F. nucleatum were inhibited by arginine and other compounds characterized by guanido groups. However, we did not find an inhibitory effect of histidine or sodium chloride on the binding F. nucleatum to sHAP, although L-arginine showed slight inhibitory activity. These findings suggest that the binding does not involve non-specific electrostatic or ionic interactions; however, it might occur through protein–protein interaction.

To determine directly the binding region of statherin to F. nucleatum cells, a dot blot assay was performed between F. nucleatum cells and peptide fragments of statherin. Following tri-partition of statherin (peptides 1–14, 14–26 and 26–43) peptide 1–14 displayed stronger ability to bind F. nucleatum cells relative to the other two peptides; however, this binding ability did not differ significantly among the three peptides. Furthermore, seven analogous peptide fragments of statherin (peptides 1–6, 6–14, 14–21, 19–26, 26–34, 32–39 and 38–43) could not bind the membrane at sufficient levels, probably due to decreased hydrophobicity of the peptides. Consequently, the binding region of statherin was examined via an inhibition assay employing the same peptide fragments.

Among peptides 1–14, 14–26, 26–43 and whole statherin as inhibitors, peptide 14–26 demonstrated the most intensive inhibitory activity; in contrast, whole statherin exerted no inhibitory effect (Fig. 2). Several reports have suggested that binding sites of salivary proteins including statherin and PRP1 in oral bacteria might be cryptic (Gibbons et al., 1991; Amano et al., 1996a; Kataoka et al., 1997; Niemi & Johansson, 2004). PRP1, in solution, showed no inhibitory effect on adhesion of Streptococcus gordonii cells (Gibbons et al., 1991) and P. gingivalis fimbriae (Kataoka et al., 1997) to PRP 1-coated HAP. In addition, the adhesion of P. gingivalis fimbriae (Amano et al., 1996a) and the cells of Actinomyces spp. (Niemi & Johansson, 2004) was not affected by statherin in solution. Domains of statherin involved in interaction with F. nucleatum are also thought to be cryptic. Among seven analogous peptidic fragments, peptides 19–26 and 32–39, which represent the middle and C-terminal regions of statherin, respectively, exhibited strong inhibitory activity. Adhesion of bacteria including Actinomyces spp. (Niemi & Johansson, 2004) and P. gingivalis (Amano et al., 1994) to the middle and C-terminal regions of statherin has been reported. Although these investigations suggested that the C-terminal residues represented by the tripeptide YTF were crucial for bacterial binding, the peptide 38–43 did not show an inhibitory effect on the binding in this study. Peptides 1–6 and 6–14 of the N-terminal fragments failed to display inhibitory activity. The N-terminal acidic region of statherin is thought to function as an anchor to HAP (Ramasubbu et al., 1993; Long et al., 2001), and therefore this region would not participate in binding of bacterial adhesins.

Furthermore, evaluation of the active segments of statherin necessary for binding of F. nucleatum cells revealed that YQPVPE (aa 21–26) and PYQPQYQ (aa 33–39) are minimal active segments (Figs 3 and 5). The YQP moiety was common to both these peptides. Amano et al. (1996a) previously suggested that LY (aa 29–30) and YTF (aa 41–43) of statherin are important regions with respect to binding of fimbriae of P. gingivalis to the statherin molecule. More recently, Niemi & Johansson (2004) reported, using a hybrid peptide construct of statherin, that some types of Actinomyces spp. bound QQYTF and PYQPYQ peptide, but other types bound YQPVPE and QPLYPQ. A Tyr residue, which exhibits hydrophobic properties and hydrogen bonding, was common to these sequences. However in this study, AQPVPE (where the tyrosine residue was replaced by alanine) retained inhibitory activity (Fig. 4), suggesting that the Tyr residue may not be important for binding. In the QP moiety, amidic Gln is characterized by hydrophilicity and strong hydrogen bonding and Pro is characterized by close involvement in higher-order structure. In addition, Gibbons et al. (1991) reported that S. gordonii may recognize a QP segment of PRP1. The moiety of the QP dipeptide may therefore play a critical role in terms of expression of binding ability. Among the findings related to inhibitory effect of alanine-scan peptides, only YAPVPE demonstrated significantly weaker inhibitory activity in comparison to YQPVPE (Fig. 4). Therefore, these data suggest that Gln may be a key amino acid in the minimum active domain of statherin.

Ramasubbu et al. (1993) proposed, via a schematic model of the structural characteristics of statherin, that in solution a dimer of statherin is formed with the clustered negatively charged N-terminal groups completely exposed for binding to enamel; however, the potential site for adhesion to bacteria is masked as a result of the folded conformation. The dimer is separated into monomers when binding to enamel, which utilizes the N-terminal helix and poly-L-proline helix; furthermore, the potential binding sites of statherin, recognized by adhesive bacteria, are exposed. More recently, dynamic NMR studies have demonstrated that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of statherin domain are mobile and weakly interact with the mineral surface (Long et al., 2001). The proposed functional sites that we found to bind F. nucleatum cells reside in the middle and C-terminal portions of statherin. This schematic model can explain the molecular mechanism by which the binding sites located in the middle and the C-terminal portion of soluble statherin are concealed. The present study determined the binding sites of F. nucleatum strains to sHAP via inhibition employing synthetic fragments of statherin. Additional evidence to support the understanding of the binding mechanism, including correlation of the three-dimensional structure with the binding sites of statherin to F. nucleatum, may be obtained by NMR, FT-IR and X-ray analyses.


   ACKNOWLEDGEMENTS
 
This study was supported in part by Grants-in-Aid (A-11771315, C-13672145) from the Ministry of Education, Science, Sports and Culture of Japan, and in part by Grants of the 21st Century COE entitled ‘Origination of Frontier BioDentistry’ at Osaka University Graduate School of Dentistry, supported by the Ministry of Education, Culture, Sports, Science and Technology.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aimoto, S. (1989). Chemical synthesis of peptide and protein, present and perspective. Seikagaku 61, 300–303.[Medline]

Amano, A., Sojar, H. T., Lee, J.-Y., Sharma, A., Levine, M. J. & Genco, R. J. (1994). Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis. Infect Immun 62, 3372–3380.[Abstract]

Amano, A., Kataoka, K., Raj, P. A., Genco, R. J. & Shizukuishi, S. (1996a). Binding sites of salivary statherin for Porphyromonas gingivalis recombinant fimbrillin. Infect Immun 64, 4249–4254.[Abstract]

Amano, A., Sharma, A., Lee, J.-Y., Sojar, H. T., Raj, P. A. & Genco, R. J. (1996b). Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary proline-rich protein and statherin. Infect Immun 64, 1631–1637.[Abstract]

Bradshaw, D. J., Marsh, P. D., Watson, G. K. & Allison, C. (1998). Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration. Infect Immun 66, 4729–4732.[Abstract/Free Full Text]

Dehazya, P. & Coles, R. S., Jr (1980). Agglutination of human erythrocytes by Fusobacterium nucleatum; factors influencing hemagglutination and some characteristics of the agglutinin. J Bacteriol 143, 205–211.[Medline]

Falkler, W. A., Jr & Burger, B. W. (1981). Microbial surface interactions; reduction of the hemagglutination activity of the oral bacterium Fusobacterium nucleatum by absorption with Streptococcus and Bacteroides. Arch Oral Biol 26, 1015–1025.[CrossRef][Medline]

Falkler, W. A., Jr, Smoot, C. N. & Mongiello, J. R. (1982). Attachment of cell fragments of Fusobacterium nucleatum to oral epithelial cells, gingival fibroblasts and white blood cells. Arch Oral Biol 27, 553–559.[CrossRef][Medline]

Gibbons, R. J. & Hay, D. I. (1988). Human salivary acidic proline-rich proteins and statherin promote attachment of Actinomyces viscosus LY7 to apatitic surfaces. Infect Immun 56, 439–445.[Medline]

Gibbons, R. J., Hay, D. I., Childs, W. C. III. & Davis, G. (1990). Role of cryptic receptors (Cryptitopes) in bacterial adhesion to oral surfaces. Arch Oral Biol 35, 107S–114S.[CrossRef][Medline]

Gibbons, R. J., Hay, D. I. & Schlesinger, D. H. (1991). Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces. Infect Immun 59, 2948–2954.[Medline]

Han, Y. W., Shi, W., Huang, G. T. J., Haake, S. K., Park, N. H., Kuramitsu, H. & Genco, R. J. (2000). Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect Immun 68, 3140–3146.[Abstract/Free Full Text]

Hay, D. I. & Moreno, E. C. (1989). Statherin and the acidic proline-rich proteins. In Human Saliva: Clinical Chemistry and Microbiology, pp. 131–150. Edited by J. Tenovuo. Boca Raton, FL: CRC Press.

Johnsson, M., Levine, M. J. & Nancollas, G. H. (1993). Hydroxyapatite binding domains in salivary proteins. Crit Rev Oral Biol Med 4, 371–378.[Medline]

Kataoka, K., Amano, A., Kuboniwa, M., Horie, H., Nagata, H. & Shizukuishi, S. (1997). Active sites of salivary proline-rich protein for binding to Porphyromonas gingivalis fimbriae. Infect Immun 65, 3159–3164.[Abstract]

Kataoka, K., Amano, A., Kawabata, S., Nagata, H., Hamada, S. & Shizukuishi, S. (1999). Secretion of functional salivary peptide by Streptococcus gordonii which inhibits fimbria-mediated adhesion of Porphyromonas gingivalis. Infect Immun 67, 3780–3785.[Abstract/Free Full Text]

Kaufman, J. & DiRienzo, J. M. (1988). Evidence for the existence of two classes of corncob (coaggregation) receptor in Fusobacterium nucleatum. Oral Microbiol Immunol 3, 145–152.[Medline]

Kolenbrander, P. E. & Andersen, R. N. (1989). Inhibition of coaggregation between Fusobacterium nucleatum and Porphyromonas (Bacteroides) gingivalis by lactose and related sugars. Infect Immun 57, 3204–3209.[Medline]

Kolenbrander, P. E. & London, J. (1993). Adhere today, here tomorrow: oral bacterial adherence. J Bacteriol 175, 3247–3252.[Medline]

Kuboniwa, M., Amano, A. & Shizukuishi, S. (1998). Hemoglobin-binding protein purified from Porphyromonas gingivalis is identical to lysine-specific cysteine proteinase Lys-gingipain. Biochem Biophys Res Commun 249, 38–43.[CrossRef][Medline]

Lee, J. Y., Sojar, H. T., Bedi, G. S. & Genco, R. J. (1992). Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. Infect Immun 60, 1662–1670.[Abstract]

Long, J. R., Shaw, J. W., Stayton, S. P. & Drobny, P. G. (2001). Structure and dynamics of hydrated statherin on hydroxyapatite as determined by solid-state NMR. Biochemistry 40, 15451–15455.[CrossRef][Medline]

Murray, P. A., Kern, D. G. & Winkler, J. R. (1988). Identification of a galactose-binding lectin on Fusobacterium nucleatum FN-2. Infect Immun 56, 1314–1319.[Medline]

Niemi, D. L. & Johansson, I. (2004). Salivary statherin peptide-binding epitopes of commensal and potentially infectious Actinomyces spp. delineated by a hybrid peptide construct. Infect Immun 72, 782–787.[Abstract/Free Full Text]

Ramasubbu, N., Reddy, M. S., Bergey, E. J., Haraszthy, G. G., Soni, S. D. & Levine, M. J. (1991). Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem J 280, 341–352.[Medline]

Ramasubbu, N., Thomas, L. M., Bhandray, K. K. & Levine, M. J. (1993). Structural characteristics of human salivary statherin: a model for boundary lubrication at the enamel surface. Crit Rev Oral Biol Med 4, 363–370.[Abstract]

Scannapieco, F. A. (1994). Saliva-bacterium interactions in oral microbial ecology. Crit Rev Oral Biol Med 5, 203–248.[Abstract]

Strömberg, N., Borén, T., Carlén, A. & Olsson, J. (1992). Salivary receptors for GalNAc{beta}-sensitive adherence of Actinomyces spp: evidence for heterogeneous GalNAc{beta} and proline-rich protein receptor properties. Infect Immun 60, 3278–3286.[Abstract]

Xie, H., Gibbons, R. J. & Hay, D. I. (1991). Adhesive properties of strains of Fusobacterium nucleatum of the subspecies nucleatum, vincentii and polymorphum. Oral Microbiol Immunol 6, 257–263.[Medline]

Yao, Y., Grogan, J., Zehnder, M., Lendenmann, U., Nam, B., Wu, Z., Costello, C. E. & Oppenheim, F. G. (2001). Compositional analysis of human acquired enamel pellicle by mass spectrometry. Arch Oral Biol 46, 293–303.[CrossRef][Medline]

Yao, Y., Berg, E. A., Costello, C. E., Troxler, R. F. & Oppenheim, F. G. (2003). Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches. J Biol Chem 278, 5300–5308.[Abstract/Free Full Text]

Received 19 February 2004; revised 26 March 2004; accepted 26 March 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Sekine, S.
Articles by Shizukuishi, S.
Articles citing this Article
PubMed
PubMed Citation
Articles by Sekine, S.
Articles by Shizukuishi, S.
Agricola
Articles by Sekine, S.
Articles by Shizukuishi, S.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.