A Peptide Domain of Bovine Milk Lactoferrin Inhibits the Interaction between Streptococcal Surface Protein Antigen and a Salivary Agglutinin Peptide Domain

Abstract

The peptide domain of salivary agglutinin responsible for its interaction with cell surface protein antigen (PAc) of Streptococcus mutans or bovine lactoferrin was found in the same peptide, scavenger receptor cysteine-rich domain peptide 2 (SRCRP2). Inhibition studies suggest that PAc and lactoferrin, of which residues 480 to 492 seem important, competitively bind to the SRCRP2 domain of salivary agglutinin.

Streptococcus mutans has been implicated as the prime cause of dental caries, one of the most common diseases in humans (24, 25). Colonization of the tooth surface by S. mutans is initiated in part by binding of the organism to salivary components adsorbed on tooth surfaces (13). This binding is mediated by a 190-kDa cell surface protein antigen (PAc) of S. mutans, variously designated as antigen I/II, B, IF, P1, SR, MSL-1, and SpaP (3, 13, 14, 24). It has been previously shown that salivary agglutinin, a 300- to 400-kDa glycoprotein, binds to PAc (23). Several studies (11, 15, 17) have demonstrated that salivary agglutinin is a member of the scavenger receptor cysteine-rich (SRCR) superfamily. To identify the S. mutans-binding peptide domain of salivary agglutinin, Bikker et al. (2) synthesized consensus-based peptides of the SRCR domains and SRCR-interspersed domains (SIDs) and found that only one peptide, SRCR domain peptide 2 (SRCRP2), bound to S. mutans cells.

Bovine milk lactoferrin has been a widely studied protein (4, 9, 21, 27, 29) that displays various characteristics. In previous studies (16, 22), it has been shown that bovine lactoferrin inhibits both the saliva-induced S. mutans aggregation and the adherence of the organism to a salivary film by binding strongly to salivary agglutinin. By using deletion analysis, it was also demonstrated that amino acid residues 473 to 538 of lactoferrin (Lf411) are implicated in these activities (16).

In the present study, we sought to identify the agglutinin peptide domain responsible for binding to PAc and to lactoferrin. Next, the inhibitory effects of lactoferrin on the interaction between PAc and the identified peptide domain of the agglutinin were examined. By preparing several lactoferrin peptides, a short peptide domain within the Lf411 motif responsible for the interaction with the agglutinin peptide was identified.

PAc binding to salivary agglutinin peptides.

Unstimulated whole saliva was collected from a single donor (male, 45 years of age), and salivary agglutinin was isolated by using the method of Oho et al. (23). Recombinant PAc (rPAc) was purified from the supernatants of transformant S. mutans TK18 cultures (13). Agglutinin-derived peptides (2) and peptides within the Lf411 motif were synthesized by solid-phase peptide synthesis using Fmoc [N-(9-fluorenyl)methoxycarbonyl] chemistry as described previously (28). The consensus sequences of the 13 N-terminal SRCR domains and 11 SIDs of agglutinin were determined based on the amino acid sequence of gp-340 (12). Bovine lactoferrin peptides were designed and synthesized based on the amino acid sequence of the molecule (8). Peptides were purified by reverse-phase high-performance liquid chromatography with a JASCO (Tokyo, Japan) high-performance liquid chromatography system according to the method described by Bikker et al. (2). The authenticity of the peptides was confirmed by quadrupole time of flight mass spectrometry on a tandem mass spectrometer (Micromass Inc., Manchester, United Kingdom) as described previously (20). The purity of the peptides was at least 90%.

To examine the binding of PAc to agglutinin peptides, solid-phase assays were performed. Microtiter plates were coated with various amounts of synthetic peptides or purified agglutinin. The peptides tested were SRCRP1, SRCRP2, SRCRP3, SRCRP4, SRCRP5, SRCRP6, SRCRP7, and SID22 (2) (Table 1). The peptides and agglutinin were dissolved in coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) to an initial concentration of 40 μg/ml and diluted serially, and then 100 μl of each solution was added to the plate. After incubation at 37°C for 90 min, the plate was washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST). Subsequently, 100 μl of biotinylated rPAc (5 μg/ml in PBST) was added and incubated at 37°C for 90 min. To examine the calcium specificity of the PAc binding to agglutinin peptides, 1 mM CaCl₂ or 1 mM EDTA was added to PBST. Plates were washed three times with PBST, and the bound biotinylated rPAc was detected with alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, Calif.), followed by the addition of p-nitrophenylphosphate substrate solution (1 mg/ml). After a 30-min incubation at 37°C, the A₄₀₅ was measured with a microplate reader (Bio-Rad Laboratories, Richmond, Calif.).

TABLE 1.

Peptides used in this study that are derived from salivary agglutinin and lactoferrin fragment Lf411

Substance and derived peptide	Amino acid sequence	No. of amino acids
Agglutinin
SRCRP1	GSESSLALRLVNGGDRC	17
SRCRP2	QGRVEVLYRGSWGTVC	16
SRCRP3	DDSWDTNDANVVCRQLGC	18
SRCRP4	GWAMSAPGNARFGQGSGPIVLDDVRC	26
SRCRP5	SGHESYLWSC	10
SRCRP6	PHNGWLSHNC	10
SRCRP7	GHHEDAGVICSA	12
SID22	AQSWSTPRPDTLPTITLPASTV	22
Lactoferrin
Lf(480-492)a	SCAFDEFFSQSCA	13
Lf(490-502)	SCAPGADPKSRLC	13
Lf(499-511)	SRLCALCAGDDQG	13
Lf(519-532)	SKEKYYGYTGAFRC	14

^aThe numbers in the parentheses are amino acid positions according to Goodman and Schanbacher (8).

As a result, rPAc bound to only one of the eight peptides tested, i.e., SRCRP2 (Fig. 1A). Preincubations of rPAc with various concentrations of SRCRP2 showed that rPAc binding to agglutinin was inhibited in a dose-dependent manner, suggesting that the binding sites of PAc for SRCRP2 and agglutinin are identical or at least located in close proximity (Fig. 2). The calcium specificity for the reaction was also examined. The binding of rPAc to SRCRP2 was strong even in the presence of EDTA, whereas the binding of rPAc to immobilized salivary agglutinin required the presence of a calcium ion (Table 2). It has been reported that the interaction between salivary agglutinin and S. mutans cells (and thus presumably PAc) is calcium dependent (6, 23). In the present study, however, the binding of rPAc to SRCRP2 occurred in the presence of EDTA, indicating that the interaction between PAc and the peptide requires no calcium ions. Calcium ions have been reported to play a role in conformational changes within protein molecules that result in the exposure of ligand-binding sites (7, 18). The results obtained in this study suggest that calcium ions might be required for the conformational change in salivary agglutinin to expose the SRCRP2 peptide domain for PAc binding.

FIG. 1.

Binding of rPAc (A) and lactoferrin (B) to agglutinin peptides. The binding is expressed as the A₄₀₅ values obtained by using peptide-coated microplates (20 μg/ml).

FIG. 2.

Inhibitory effect of SRCRP2 on the binding of rPAc to salivary agglutinin. The binding is expressed as the A₄₀₅ values obtained by using agglutinin-coated microplates (5 μg/ml). Values are given as the means ± standard deviations of triplicate assays.

TABLE 2.

Effects of calcium ions on the binding of rPAc to SRCRP2 and purified agglutinin

Substance tested	Bindinga of rPAc in the presence of:
	No additives	CaCl2	EDTA
SRCRP2	1.13 ± 0.10	1.04 ± 0.01	1.08 ± 0.09
Agglutinin	0.14 ± 0.04	1.05 ± 0.02	0.20 ± 0.06

^aBinding is expressed as the A₄₀₅ value obtained with SRCRP2-coated (20 μg/ml) and agglutinin-coated (5 μg/ml) microplates. The values are the means ± standard deviations of triplicate assays.

Lactoferrin binding to salivary agglutinin peptides.

The binding of lactoferrin to agglutinin peptides was also examined by solid-phase assays as described above. Bovine lactoferrin was purchased from Sigma Chemical Co. (St. Louis, Mo.). One hundred microliters of biotinylated lactoferrin (5 μg/ml in PBST) was added to the peptide-coated microplates. Incubations of lactoferrin with the SRCR and SID peptides identified SRCRP2 as the lactoferrin-binding peptide (Fig. 1B). The specificity of lactoferrin binding to SRCRP2 was also confirmed with agglutinin-coated microplates (data not shown).

For inhibition studies, lactoferrin, lactoferrin fragment Lf411, or several synthetic peptides derived from the Lf411 sequence were added to the biotinylated rPAc before application to the assay plates. The lactoferrin peptides used are listed in Table 1. Inhibition studies with lactoferrin and rPAc established competitive binding for both ligands of SRCRP2 (Fig. 3). Lactoferrin fragment Lf411, which has been shown to inhibit S. mutans binding to salivary agglutinin, also inhibited rPAc binding to SRCRP2 in a dose-dependent manner. To identify the binding region of lactoferrin to SRCRP2 at the peptide level, four peptides derived from Lf411 were synthesized. One peptide, i.e., the peptide at amino acids 480 to 492 of lactoferrin [Lf(480-492)], strongly inhibited the rPAc binding to SRCRP2, whereas other peptides showed almost no inhibition (Fig. 4).

FIG. 3.

Inhibitory effect of lactoferrin on the binding of rPAc to SRCRP2. The binding is expressed as the A₄₀₅ values obtained by using SRCRP2-coated microplates (20 μg/ml). Values are given as the means ± standard deviations of triplicate assays.

FIG. 4.

Inhibitory effect of lactoferrin peptides derived from Lf411 on the binding of rPAc to SRCRP2. Binding is expressed as the A₄₀₅ values obtained by using SRCRP2-coated microplates (20 μg/ml). The concentration of each lactoferrin peptide used was 12.5 μg/ml. The percent inhibition was calculated as follows: percent inhibition = 100 × [(a − b)/a], where a is the mean A₄₀₅ without an inhibitor and b is the mean A₄₀₅ with an inhibitor (lactoferrin peptide).

Salivary agglutinin, recently identified as a member of the SRCR superfamily (11, 15), has been thought to be involved in the initiation of dental caries, since it is part of the pellicle and stimulates the attachment of S. mutans cells (5, 26). One of the surface components of S. mutans, PAc, is a ligand for salivary agglutinin (10, 19), and Bikker et al. (2) demonstrated that S. mutans cells interact with only one of the SRCR peptides, SRCRP2, of salivary agglutinin. However, the interaction between the bacterial surface protein and the peptide domain of salivary agglutinin has not yet been clarified. In this study, we have used the various agglutinin peptides, together spanning the complete consensus binding sequence, in a binding assay and showed that rPAc bound only to SRCRP2. This finding supports the data of Bikker et al. (2) and confirms the role of PAc as a bacterial ligand for the agglutinin. We also showed that lactoferrin, like rPAc, bound only to SRCRP2. More interestingly, lactoferrin inhibited the binding of rPAc to SRCRP2, suggesting that bovine lactoferrin plays a role in the defense against oral colonization by S. mutans. Lactoferrin fragment Lf411 also inhibited rPAc binding to SRCRP2. Furthermore, one particular peptide motif, i.e., Lf(480-492), in the Lf411 domain seemed to contain the inhibitory activity. In the next step, it is necessary to establish the inhibitory effect of the Lf(480-492) peptide on the binding of PAc or S. mutans cells to agglutinin-coated tooth surfaces.

The binding mechanism of SRCRP2 to rPAc and lactoferrin or the Lf(480-492) peptide remains unclear. Further studies are necessary to elucidate this mechanism by using scrambled peptides of SRCRP2 and Lf(480-492). Human saliva also contains lactoferrin, and it would be favorable if it could inhibit the binding of S. mutans cells to salivary agglutinin as an innate factor. However, since human lactoferrin does not contain exactly the same sequence as that of Lf(480-492) derived from bovine lactoferrin, the inhibitory effect is uncertain. In addition, lactoferrin is an iron-binding protein, and its iron-binding capacity is associated with biological functions (1). However, there are no iron-binding sites within the residues 480 to 492 of bovine lactoferrin (8), and the binding of Lf(480-492) to SRCRP2 seems to be independent of the presence of iron ions.

In conclusion, we demonstrated that both the rPAc of S. mutans and bovine lactoferrin bind to peptide domain SRCRP2 of salivary agglutinin and that lactoferrin inhibits the interaction between rPAc and SRCRP2. The lactoferrin residues 480 to 492 are important for this inhibition, and the peptide corresponding to this region could potentially be used to inhibit the adherence of S. mutans cells to salivary films.