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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 114, 1426, 2643, 16, 614, 1421, 1926, 2634, 3239 and 3843) corresponding to the amino acid sequence of statherin were synthesized commercially (Table 1
, Kataoka et al., 1997
). Peptides 114 (16 and 614) corresponded to the N-terminal helix domain; peptides 1426 (1421 and 1926) were contained in the middle region of statherin; and peptides 2643 (2634, 3239 and 3843) 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).
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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 ml1) 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 ml1) 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 ml1) 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 ml1) 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.
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RESULTS |
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Determination of the binding regions of statherin utilizing analogous peptides
Three synthetic peptides corresponding to amino acid residues 114, 1426 and 2643 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 1426 displayed significant inhibition of 48 %; peptide 2643 inhibited whole-cell binding by 38 %, whereas the inhibitory effect of peptide 114 was 26 %.
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Determination of the minimal active segments in residues 1926 (GPYQPVPE) and 3239 (QPYQPQYQ)
Serial peptides, in which amino acid residues were deleted from the N-terminus of peptide 1926 and from the C-terminus of peptide 2126, were synthesized to identify the minimal active segment of peptide 1926 (Fig. 3). Serial deletions of the Gly and Pro residues in peptide 1926 (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 2126 (YQPVPE).
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DISCUSSION |
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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 proteinprotein 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 114, 1426 and 2643) peptide 114 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 16, 614, 1421, 1926, 2634, 3239 and 3843) 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 114, 1426, 2643 and whole statherin as inhibitors, peptide 1426 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 1926 and 3239, 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 3843 did not show an inhibitory effect on the binding in this study. Peptides 16 and 614 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 2126) and PYQPQYQ (aa 3339) 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 2930) and YTF (aa 4143) 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 19 February 2004;
revised 26 March 2004;
accepted 26 March 2004.
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