inhibitory effect of bovine milk lactoferrin on the ... · bovine lactoferrin to salivary...

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Inhibitory Effect of Bovine Milk Lactoferrin on the Interaction between a Streptococcal Surface

Protein Antigen and Human Salivary Agglutinin*

Morihide Mitoma, Takahiko Oho‡, Yoshihiro Shimazaki, and Toshihiko Koga

From the Department of Preventive Dentistry, Kyushu University Faculty of Dental Science, Fukuoka

812-8582, Japan

* This work was supported in part by Grants-in-Aid for Developmental Scientific Research

(A)12357013 (T.K.) and (C) 11672051 (T.O.) from the Ministry of Education, Science, Sports and

Culture of Japan and by the Kyushu University Interdisciplinary Programs in Education and Projects in

Research Development (T.K.).

‡ To whom correspondence should be addressed. Tel.: +81-92-642-6353; Fax: +81-92-642-6354;

E-mail: [email protected]

Running Title: Binding of bovine lactoferrin to salivary agglutinin

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 13, 2001 as Manuscript M101459200 by guest on Septem

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SUMMARY

Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between

a surface protein antigen (PAc) of the organism and salivary agglutinin. Bovine milk inhibits the saliva-

induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity

against this aggregation was isolated and found to be lactoferrin. Surface plasmon resonance analysis

indicated that bovine lactoferrin binds more strongly to salivary agglutinin, especially to high-molecular-

mass glycoprotein which is a component of the agglutinin, than to recombinant PAc. The binding of

bovine lactoferrin to salivary agglutinin was thermostable and the optimal pH for binding was 4.0. To

identify the saliva-binding region of bovine lactoferrin, 11 truncated bovine lactoferrin fragments were

constructed. A fragment corresponding to the C-terminal half of the lactoferrin molecule had a strong

inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the

N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to lactoferrin

residues 473 to 538 also showed a high ability to inhibit the aggregation of S. mutans. These results

suggest that residues 473 to 538 of bovine lactoferrin are important in the inhibition of saliva-induced

aggregation of S. mutans.

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INTRODUCTION

Streptococcus mutans has been strongly implicated in causation of dental caries, a common human

disease (1, 2). Colonization of the tooth surface by S. mutans is initiated by binding of the organism to

salivary components on tooth surfaces (3). This binding is mediated by a 190-kDa surface protein

antigen (PAc1) of S. mutans, variously designed antigen I/II, B, IF, P1, SR, MSL-1 (1, 3-5). Various

salivary components have been reported to bind to S. mutans or to induce its aggregation (6-8). We have

recently shown that the PAc of S. mutans binds to a complex of high-molecular-mass salivary

glycoprotein and secretory immunoglobulin A (sIgA) (9).

Bovine milk is commonly found in the human diet. Since bovine milk is produced on a large scale

at low cost, and is easily delivered to the oral cavity, it has been used for passive immunization in

prevention measures targeting several pathogens (10-13). Bovine milk contains several protein

components, including caseins, immunoglobulins, lactalbumin, lactoferrin, lactoglobulin,

lactoperoxidase, and lysozyme (14). Casein and lactoperoxidase have been reported to inhibit the

adherence of S. mutans to saliva-coated hydroxyapatite (15, 16). κ-Casein reduces the

glucosyltransferase activity of S. mutans, which in turn reduces glucan formation (17), and lactoferrin has

a bactericidal effect on S. mutans (18).

In this study, we examined the effects of bovine milk on the saliva-induced aggregation of S.

mutans cells. We purified and characterized the aggregation-inhibitory activity present in milk, and

determined that this activity is due to lactoferrin. The interaction between lactoferrin and salivary

agglutinin was further examined by surface plasmon resonance. Finally, deletion analysis of lactoferrin

was used to identify the region of lactoferrin responsible for its interaction with saliva.

EXPERIMENTAL PROCEDURES

Bacterial Strains–––S. mutans strains MT8148 (3) and Xc (19) were used as representative strains

of S. mutans serotype c. S. mutans TK18 is a recombinant strain that produces a large amount of PAc (3).

S. sanguinis ATCC 10556, S. oralis ATCC 10557, and S. gordonii ATCC 10558 were used as type


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strains. Escherichia coli M15[pREP4] was obtained from Qiagen. The culture media used were 2 × TY

broth (20) for E. coli and brain heart infusion (BHI, Difco) broth for streptococci.

Saliva–––Unstimulated whole saliva was collected from a single donor (male, 42 years of age) in

an ice-chilled tube and clarified by centrifugation at 12,000 × g for 10 min.

Salivary Agglutinin–––Salivary agglutinin was isolated by the method of Oho et al. (9). Briefly,

clarified whole saliva diluted 1/2 with aggregation buffer (1.5 mM KH2PO4, (pH 7.2), 6.5 mM Na2HPO4,

2.7 mM KCl, 137 mM NaCl) was incubated with an equal volume of a cell suspension of S. mutans

MT8148 at 37°C for 30 min. Cells were collected by centrifugation and washed with aggregation buffer,

and adsorbed salivary agglutinin was eluted with the same buffer supplemented with 1 mM EDTA. The

eluate was filtered (0.2-µm pore size) and subjected to gel filtration chromatography on Superdex 200 HR

(Amersham Pharmacia Biotech) equilibrated with aggregation buffer. The eluate at the void volume was

collected and used as salivary agglutinin. For the surface plasmon resonance analysis to examine the

binding of lactoferrin, salivary agglutinin was dissociated into its components of high-molecular-mass

glycoprotein and sIgA by electrophoretic fractionation (9).

rPAc–––Recombinant PAc (rPAc) was purified from the culture supernatants of transformant S.

mutans TK18 by ammonium sulfate precipitation, chromatography on DEAE-cellulose, and subsequent

gel filtration on Sepharose CL-6B (Amersham Pharmacia Biotech) (3).

Milk Components–––Bovine α-casein, β-casein, κ-casein, lactalbumin, lactoferrin, and

lactoperoxidase were purchased from Sigma. Bovine γ-casein was purchased from Research Organics,

and bovine lactoglobulin from ICN Biomedicals. Bovine immunoglobulin G was prepared from bovine

milk, using affinity chromatography on a HiTrap protein G column (5 ml) (Amersham Pharmacia

Biotech) according to the method of Oho et al. (21). Iron-saturated bovine lactoferrin and iron-free

lactoferrin (apo-lactoferrin) were prepared from bovine lactoferrin according to the methods of Kawasaki

et al. (22) and Shimazaki et al. (23), respectively. The degree of iron saturation of lactoferrin was

determined by the Wako Fe-B test (Wako, Osaka, Japan). Bovine lactoferrin (Sigma) was determined to

be 19.3% iron-saturated. Lactoferricin B was a gift from the Nutrition Science Laboratory, Morinaga

Milk Industry Co., Japan. Protein content was determined according to the method of Lowry et al. (24),

with bovine serum albumin as a standard.


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Fractionation of Bovine Milk–––The milk component responsible for inhibiting aggregation was

isolated by subjecting bovine milk to fast protein liquid chromatography (FPLC). First, the milk fat was

removed by centrifugation at 12,000 × g for 15 min, and the skimmed milk was dialyzed against 10 mM

imidazole-HCl buffer, pH 7.0. Then, the milk sample was passed through a 0.2-µm filter and applied to a

Mono S HR 5/5 column (Amersham Pharmacia Biotech) that had been equilibrated with 10 mM

imidazole-HCl buffer (pH 7.0). After sample application, the column was washed with 5 volumes of the

same buffer, and the bound material was eluted with a linear gradient (0 - 1 M) of NaCl in the same

buffer. Each fraction was analyzed for protein by monitoring the absorbance at 280 nm (A280), and was

assayed for aggregation-inhibitory activity.

Sequence Determination–––The N-terminal amino acid sequence of the isolated aggregation-

inhibitory bovine milk component was determined by Edman degradation using a Shimadzu PSSQ-21

gas-phase sequencer (Kyoto, Japan).

Aggregation Assay–––Streptococcal cells were suspended in aggregation buffer at an A550 of

approximately 1.5. Either 25 µl of whole saliva or 10 µl of salivary agglutinin (0.5 mg/ml) was mixed

with 1 ml of the cell suspension and various amounts of bovine milk component, and the total volume of

the reaction mixture was adjusted to 1.5 ml with aggregation buffer. CaCl2 was added to the mixture of

salivary agglutinin at a final concentration of 1 mM. Bacterial aggregation was determined by monitoring

the change in A550 at 37°C for 2 h with a visible-UV recording spectrophotometer (Ultrospec 3000,

Amersham Pharmacia Biotech).

Binding of Bovine Lactoferrin to rPAc or Salivary Agglutinin–––Surface plasmon resonance,

which permits real-time analysis of macromolecular interactions (25), was used to examine the binding of

bovine lactoferrin to rPAc, salivary agglutinin, or to components of salivary agglutinin. Binding assays

were carried out with a BIAcore 2000 surface plasmon resonance biosensor (Amersham Pharmacia

Biotech). First, rPAc, salivary agglutinin, high-molecular-mass glycoprotein separated by electrophoretic

fractionation, and sIgA separated by electrophoretic fractionation were immobilized on

carboxymethylated, dextran-coated, gold surfaced CM5 sensor chips via primary amino group linkages

according to the method of Johnsson et al. (26). For immobilization of each protein, 35 µl of a 300 µg/ml

solution in 10 mM sodium acetate buffer (pH 4.5) was passed over the activated chip surface while


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phosphate-buffered saline (PBS) (pH 7.0) was maintained at 5 µl/min throughout the immobilizing

process. Binding of rPAc, salivary agglutinin, high-molecular-mass glycoprotein, and sIgA to the chip

surfaces occurred at 5.8 ng, 7.4 ng, 7.1 ng, and 10.9 ng per mm2, respectively. Each milk component,

diluted in an appropriate running buffer, was then passed over the immobilized surface at a flow rate of

10 µl/min. The effect of pH on the binding of bovine lactoferrin to salivary agglutinin was assayed in 10

mM potassium phosphate buffer (pH 2 to 8) containing 0.15 M NaCl. Divalent cation specificity was

examined in PBS (pH 7.0) containing 0 - 20 mM CaCl2, MgCl2, or MnCl2. The dissociation phase of

binding was initiated by the injection of the diluent buffer at 10 µl/min. All binding experiments were

performed at 25°C. The surface resonance signal in each binding cycle was expressed in resonance units

(RU). A resonance of 1,000 RU corresponds to a shift of 0.1° in the resonance angle, which corresponds

to a change in surface protein concentration of approximately 1 ng/mm2 (27).

Heat Treatment–––In thermal stability studies, lactoferrin was heated at 40°C to 100°C for 15 min

and was then subjected to the surface plasmon resonance binding assay.

Bovine Lactoferrin Fragments–––Truncated bovine lactoferrin fragments were prepared as 6×His-

tagged fusion proteins by cloning of PCR-amplified lactoferrin gene fragments into expression vector

pQE-30 (Qiagen). The following sets of primers were used for amplification: LfN-F, 5’-

TATAGAGCTCATGAAGCTCTTCGTCCCC-3’; LfN-R, 5’-

ACACGTCGACTTACCTGGTGTACCGCGCCTT-3’; LfC-F, 5’-

TATAGGATCCGTCGTGTGGTGTGCCGTG-3’; LfC-R, 5’-

ACACGTCGACTTACCTCGTCAGGAAGGCGCA-3’; Lf4-R, 5’-

ACACGTCGACTTACAACCTGAAGTCCTCACG-3’; Lf41-R, 5’-

ACACGTCGACTTACCCAACGTCCTCAGCCAG-3’; Lf42-R, 5’-

ACACGTCGACTTAACACAAGGCACAGAGTCT-3’; Lf43-R, 5’-

ACACGTCGACTTAGCCCATGGGGATGTTCCA-3’; Lf44-R, 5’-

ACACGTCGACTTAGACAACTGCCACGGCAAG-3’; Lf45-F, 5’-

TATAGGATCCGGCCAGAACGTGACCTGT-3’; Lf46-F, 5’-

TATAGGATCCATCTACACTGCGGGCAAG-3’; Lf47-F, 5’-

TATAGGATCCGGGTACCTTGCCGTGGCA-3’; Lf411-F, 5’-


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TATAGGATCCCTGATCGTCAACCAGACA-3’. The amplified DNAs were digested with either

BamHI and SalI, or SacI and SalI (LfN only) restriction sites (underlined) and inserted into the BamHI-

SalI or SacI-SalI sites of the pQE-30 plasmid. The ligated DNAs were then transformed into E. coli

M15[pREP4]. The truncated lactoferrin fragments (amino acid position and primer used) are LfN (amino

acid position, 1-344; primers, LfN-F and LfN-R), LfC (amino acid position, 345-689; primers, LfC-F and

LfC-R), Lf4 (amino acid position, 345-571; primers, LfC-F and Lf4-R), Lf41 (amino acid position, 345-

538; primers, LfC-F and Lf41-R), Lf42 (amino acid position, 345-505; primers, LfC-F and Lf42-R), Lf43

(amino acid position, 345-472; primers, LfC-F and Lf43-R), Lf44 (amino acid position, 345-439; primers,

LfC-F and Lf44-R), Lf45 (amino acid position, 366-571; primers, Lf45-F and Lf4-R), Lf46 (amino acid

position, 399-571; primers, Lf46-F and Lf4-R), Lf47 (amino acid position, 432-571; primers, Lf47-F and

Lf4-R), and Lf411 (amino acid position, 473-538; primers, Lf411-F and Lf41-R). As a control, 6×His-

tagged mouse dihydrofolate reductase (DHFR) fusion protein was produced. Expression vector pQE-40

(Qiagen) which contains DNA fragment encoding the DHFR was transformed into E. coli M15[pREP4].

Lactoferrin and DHFR fusion proteins were extracted from whole cell extracts of E. coli

M15[pREP4] cells containing the recombinant plasmids. Cells were cultured in 2 × TY broth containing

100 µg/ml ampicillin and 25 µg/ml kanamycin at 37°C until an A550 of 1.0 was attained. Expression was

induced by addition of isopropyl-β-D(-)-thiogalactopyranoside to the cultures at a final concentration of 1

mM, and the cultures were grown for 3 h. Cells were harvested by centrifugation at 5,000 × g for 20 min,

and one-step purification of the fusion proteins was performed with Ni2+-HiTrap chelating columns (1 ml)

(Amersham Pharmacia Biotech) according to the manufacture’s instructions. In brief, the cell pellet was

solubilized in 10 mM Tris -HCl (pH 8.0), 0.1 M sodium phosphate, 6 M guanidine-HCl (buffer A) at 5

ml/g and mixed by inversion for 1 h at 4°C. The lysate was centrifuged at 10,000 × g for 20 min at 4°C,

and the cleared supernatant was applied to a Ni2+-HiTrap chelating column that had been equilibrated

with buffer A. The column was extensively washed with buffer A and then with 5 or more volumes of 10

mM Tris -HCl (pH 8.0), 0.1 M sodium phosphate, 8 M urea (buffer B) containing 10 mM imidazole until

the A280 of eluant was less than 0.01. The fusion proteins were eluted with buffer B containing 250 mM

imidazole.


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The eluted proteins were refolded by sequential dialysis against buffers containing decreasing urea

concentrations for 18 h in each buffer at 4°C (28). The gradient buffers contained 4, 2, and 1M urea in

0.1 M Tris -HCl (pH 8.0), 0.1 M sodium phosphate, and 2 mM dithiothreitol. After dialysis against 1 M

urea, fusion proteins were dialyzed against 50 mM sodium phosphate (pH 8.0) containing 0.3 M NaCl for

18 h at 4°C. Each fusion protein was analyzed by SDS-PAGE.

SDS-PAGE and Western Blotting–––SDS-PAGE was performed using 12.5% and 15%

polyacrylamide gels according to the method of Laemmli (29). After electrophoresis, the gels were

stained with Coomassie brilliant blue R-250. Electrophoresis calibration kits (Amersham Pharmacia

Biotech) were used as molecular mass markers. For Western blotting, samples were subjected to SDS-

PAGE and transferred electrophoretically to nitrocellulose membranes according to the method of

Burnette (30). After blocking with 1% bovine serum albumin in Tris -buffered saline (20 mM Tris -HCl

(pH 7.5), 150 mM NaCl) containing 1% Triton X-100, the membranes were treated with alkaline

phosphatase-conjugated goat anti-bovine lactoferrin antiserum (Betchyl Laboratories).

Statistical Analysis–––Differences between the control and the test samples in aggregation were

determined by Student’s t test.

RESULTS

Isolation and Characterization of the Milk Component that Inhibits Aggregation–––The FPLC

fraction of bovine milk eluted at 0.64 M NaCl inhibited the saliva-induced aggregation of S. mutans cells

(Fig. 1). Coomassie staining of the SDS gel revealed a single 80-kDa band in this fraction (Fig. 2A, lane

2). In Western blot, rabbit anti-bovine lactoferrin antiserum reacted with this band (Fig. 2B, lane 1). The

N-terminal amino acid sequence of this component was Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr,

which corresponds to the N-terminus of bovine lactoferrin (31). These results indicated that the

aggregation-inhibitory component is lactoferrin.

Aggregation of Streptococcal Cells–––Aggregation of the typical S. mutans strain MT8148

(serotype c) in the presence of whole saliva or salivary agglutinin was examined by a spectrophotometric


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assay. Both whole saliva and salivary agglutinin induced strong aggregation. Testing of various bovine

milk components revealed that lactoferrin inhibited this saliva-induced aggregation in a dose-dependent

manner (Fig. 3). Of the milk components tested, bovine lactoferrin had the strongest inhibitory activity,

whereas other components, such as lactoperoxidase, α-casein, and κ-casein, showed weak inhibitory

activity (Table I). Other oral streptococci, such as S. mutans Xc, S. sanguinis ATCC 10556, S. oralis

ATCC 10557, and S. gordonii ATCC 10558, were also tested for their ability to aggregate in the presence

of whole saliva with or without bovine lactoferrin. Bovine lactoferrin showed the same inhibitory effect

on the aggregation of these strains that it did on the aggregation of S. mutans MT8148 (Table II).

Binding of Bovine Lactoferrin to rPAc or Salivary Agglutinin–––The binding of bovine lactoferrin

to rPAc, salivary agglutinin, or to components of salivary agglutinin separated by electrophoretic

fractionation was analyzed by surface plasmon resonance. Lactoferrin (50 µg/ml) in PBS (pH 7.0) was

allowed to react with immobilized ligands on a sensor chip. The biosensor response of bovine lactoferrin

to rPAc, salivary agglutinin, high-molecular-mass glycoprotein, and sIgA was 149 ± 16, 470 ± 13, 718

± 47, and 34 ± 1 RU/ng of immobilized ligand, respectively (mean ± S.D. of triplicate assays).

Binding of bovine lactoferrin to immobilized salivary agglutinin was enhanced by the addition of

CaCl2 to the running buffer, with an optimum concentration of 0.5 mM CaCl2. MgCl2 and MnCl2 did not

enhance binding (data not shown). In thermal stability studies, the biosensor response induced by binding

of bovine lactoferrin to immobilized salivary agglutinin gradually decreased as the temperature used to

heat the lactoferrin was raised. However, lactoferrin still bound to salivary agglutinin even after heating

at 100°C (Fig. 4A). The pH maximum for binding of bovine lactoferrin to salivary agglutinin was pH 4.0,

and no detectable binding occurred at pH 2.0 (Fig. 4B).

Effects of Lactoferrin Fragments on the Aggregation of S. mutans Cells–––To identify the saliva-

binding region of the bovine lactoferrin molecule, 11 6×His-tagged lactoferrin fragments were cloned and

expressed in E. coli. These fusion proteins were purified and used in spectrophotometric aggregation

assays. SDS-PAGE analysis of each lactoferrin fragment showed single band (data not shown). The N-

terminally truncated lactoferrin fragment, LfC (residues 345 to 689) strongly inhibited saliva-induced

aggregation of S. mutans cells, whereas the C-terminally truncated fragment LfN (residues 1 to 344)

weakly inhibited the aggregation (Fig. 5). Fragments, Lf4 (residues 345 to 571), Lf41 (residues 345 to


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538), Lf45 (residues 366 to 571), Lf46 (residues 399 to 571), and Lf47 (residues 432 to 571) also

exhibited strong inhibition of saliva-induced aggregation of S. mutans, as did the shorter fragment Lf411

(residues 473 to 538). In contrast, fragments Lf43 (residues 345 to 472) and Lf44 (residues 345 to 439)

exhibited only weak inhibitory activity. The 6×His-tagged DHFR, which was used as control, also

weakly inhibited aggregation.

DISCUSSION

Human saliva induces aggregation of S. mutans via an interaction between PAc of the organism

and salivary agglutinin, which is a complex of high-molecular-mass glycoprotein and sIgA (3, 9). Gong

et al. (32) also showed that salivary film on hydroxyapatite contains a complex of macromolecular protein

enriched in sIgA and α-amylase, which forms a S. sanguinis-binding site. In this study, we showed that

bovine milk lactoferrin inhibited the saliva-induced aggregation of S. mutans cells. The binding of bovine

lactoferrin to rPAc, salivary agglutinin, and components of salivary agglutinin was examined using

surface plasmon resonance. Bovine lactoferrin bound more strongly to salivary agglutinin, especially to

high-molecular-mass glycoprotein, than to rPAc, suggesting that bovine lactoferrin may inhibit the

interaction between PAc and salivary agglutinin by binding to high-molecular-mass glycoprotein of

salivary agglutinin. Aggregation of other streptococcal cells induced by whole saliva was also inhibited

by bovine lactoferrin, indicating that the inhibitory effect of lactoferrin is not specific for S. mutans.

The optimal pH for the binding of bovine lactoferrin to salivary agglutinin was 4.0, and the

stability of lactoferrin to bind to salivary agglutinin was not affected by previous heat treatment. The

isoelectric point of bovine lactoferrin is approximately 8.0 (33). It can be sterilized at high temperatures

at pH 4.0 without any significant loss of bactericidal activity, suggesting that it is thermally stable at pH

4.0 (34). Bovine lactoferrin may adopt a conformation suitable for interaction with salivary agglutinin at

this pH as well.

Lactoferrin is an iron-binding glycoprotein, and its iron-binding capacity is associated with many

biological functions (35, 36). The lactoferrin preparation used in this study was 19.3% iron-saturated. To


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examine the role of iron-binding in inhibition of S. mutans aggregation, we also prepared apo-lactoferrin

and iron-saturated lactoferrin and assayed them for their ability to inhibit the saliva-induced aggregation.

No significant differences were observed among the inhibitory properties of these three types of

lactoferrin (data not shown). These results are consistent with those of Soukka et al. (37), who observed

that these three types of lactoferrin cause no difference in the binding of S. mutans, though the assay was

performed using saliva-coated hydroxyapatite. These results suggest that iron ion in lactoferrin does not

play a significant role in the binding of bovine lactoferrin to salivary agglutinin. In another experiment,

Soukka et al. (38) showed that apo-lactoferrin effectively agglutinates S. mutans cells but not the other

bacteria. However, our preliminary study have shown that all of the three types of lactoferrin did not

induce the aggregation of S. mutans cells (39). The cause of this discrepancy may be ascribed to

differences in strain of S. mutans used or the experimental condition.

To identify the saliva-binding region of the lactoferrin molecule, we prepared a series of truncated

lactoferrin fragments and assayed their effects on the saliva-induced aggregation of S. mutans cells. Our

results suggests that lactoferrin residues 473 to 538 play an important role in the inhibition of saliva-

induced aggregation of S. mutans. Other fragments lacking these residues, such as LfN (residues 1 to

344), Lf43 (residues 345 to 472), and Lf44 (residues 345 to 439), exhibited only weak inhibitory activity.

The lactoferrin molecule is proposed to consist of two lobes (N-lobe and C-lobe) (40). The N-lobe

contains the active domains for bactericidal action and heparin-binding (31, 41), whereas the C-lobe

contains a functional domain for hepatocyte binding and internalization (42). In these previous studies,

lactoferrin fragments were prepared by tryptic cleavage of lactoferrin and isolated by high performance

liquid chromatography. Here, we prepared truncated lactoferrin fragments using recombinant DNA

technology. Our results indicate that the lactoferrin domain responsible for binding to salivary agglutinin

is within the C-lobe of the protein.

The mechanism of binding of lactoferrin to salivary agglutinin remains unclear. The predicted pI

value and secondary structure of each lactoferrin fragment were obtained using the DNA software

package, DNASIS (Hitachi Software Engineering, Tokyo, Japan). Secondary structure was predicted

according to the method of Chou and Fasman (43). Although all the active fragments containing residues

473 to 538 had acidic pI values, the inactive fragment Lf44 also had an acidic pI value (pI = 5.2).


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Therefore, electrostatic interactions do not seem to be involved in agglutinin binding. Furthermore, the

inhibitory fragments of lactoferrin did not retain characteristic secondary structures. Lactoferricin B, a

25-amino acid peptide derived from the N-lobe of bovine lactoferrin, has bactericidal activity (44). The

antibacterial properties of lactoferricin B are attributed to the disruption of target cell membranes by the

basic residues arrayed along the outside of the lactoferricin B molecule (45). We found that lactoferricin

B had no inhibitory effects on the saliva-induced aggregation of S. mutans cells (data not shown). Further

studies are necessary to elucidate the mechanism by which active lactoferrin fragments inhibit the saliva-

induced aggregation of S. mutans.

There are two types of bacterial interaction with salivary components; saliva-induced bacterial

aggregation in solution phase, and bacterial adherence to salivary components adsorbed on the tooth

surface. Gibbons and Hay (46), and Raj et al. (47) reported that proline-rich proteins and statherin serve

as pellicle receptors for some of streptococcal strains, but do not induce aggregation of the organisms in

suspension. On the basis of these findings, Gibbons (48) proposed a model that an apparent

conformational change occurs when salivary components bind to hydoxyapatite, which exposes the

binding sites for bacterial adhesin. This explains the difference between bacterial aggregation and

adherence. In the present study, we found that lactoferrin in bovine milk possessed inhibitory activity

against saliva-induced aggregation of S. mutans in solution phase. Therefore, we are unable to exclude

the possibility that milk components other than lactoferrin may possess inhibitory effect on the binding of

bacterial cells to a salivary film. Further studies are necessary to clarify effects of milk components on

the adherence of bacterial cells to a salivary film.

Lactoferrin attracted a great deal of attention for its wide variety of functions (49). Lactoferrin is

viewed as a potential contributor to dental caries prevention by virtue of its inhibitory effect on the

binding of S. mutans to acquired pellicles on the tooth surface and its bactericidal action on S. mutans

(18). We have now demonstrated that bovine lactoferrin inhibits the interaction between PAc of S.

mutans and salivary agglutinin by binding strongly to salivary agglutinin. Residues 473 to 538 of bovine

lactoferrin play an important role in the interaction of lactoferrin with salivary agglutinin.


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Acknowledgments–––We thank Kei-ichi Shimazaki and Ichiro Nakamura of the Dairy Science

Laboratory, Faculty of Agriculture, Hokkaido University, Sapporo, Japan for generously providing

bovine lactoferrin cDNA.


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1The abbreviations used are: PAc, protein antigen serotype c; sIgA, secretory immunoglobulin A; BHI,

brain heart infusion; rPAc, recombinant PAc; FPLC, fast protein liquid chromatography; PBS, phosphate-

buffered saline; RU, resonance unit; PCR, polymerase chain reaction; DHFR, dihydrofolate reductase;

PAGE, polyacrylamide gel electrophoresis.


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FIGURE LEGENDS

FIG. 1. Fractionation of bovine milk by FPLC. Ten ml of defatted milk were dialyzed against 10 mM

imidazole-HCl buffer (pH 7.0) and then applied to a Mono S HR 5/5 column. The bound material was

eluted with a linear gradient of NaCl (0 - 1.0 M) in 10 mM imidazole-HCl buffer (pH 7.0). Fractions

were monitored for protein by their absorbance at 280 nm (J) and for their inhibitory effect on the

aggregation of S. mutans cells (E). — - — - —, NaCl gradient.

FIG. 2. SDS-PAGE (A) and Western blotting (B) analyses of the aggregation-inhibitory protein

purified by FPLC. A, Milk samples were suspended in SDS-PAGE reducing buffer (1% SDS, 1% 2-

mercaptoethanol) and heated at 100°C for 3 min. Samples were then subjected to SDS-PAGE (12.5%

polyacrylamide), and the gel was stained with Coomassie brilliant blue R-250. The molecular mass

markers used were α-lactalbumin (14.4 kDa), soybean trypsin inhibitor (20 kDa), carbonic anhydrase (30

kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), and phosphorylase b (94 kDa). Lanes: 1,

defatted bovine milk (5 µg); 2, the aggregation-inhibitory protein (3 µg); 3, bovine lactoferrin from

Sigma (3 µg). B, Milk proteins on the gel were electrophoretically transferred to a nitrocellulose

membrane, and the membrane was reacted with goat antiserum against bovine lactoferrin. Lanes: 1, the

aggregation-inhibitory protein (2 µg); 2, bovine lactoferrin from Sigma (2 µg).

FIG. 3. Dose-dependent inhibition of the saliva-induced aggregation of S. mutans cells by bovine

lactoferrin. S. mutans MT8148 cells grown in BHI broth were harvested and resuspended in aggregation

buffer. The suspensions were adjusted to an A550 of approximately 1.5 with aggregation buffer. The cell

suspensions (1 ml) were mixed with 25 µl of whole saliva and various amounts of lactoferrin, and the

total volume of the reaction mixture was adjusted to 1.5 ml. Aggregation was measured by the reduction

in A550 after 2 h. Percent inhibition was calculated as 100 × [(a - b)/a], where a is the mean value without

lactoferrin (control), and b is the mean value with lactoferrin. Values are given as the means ± S.D. of

triplicate assays.


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FIG. 4. Heat stability of bovine lactoferrin (A) and the effect of pH on the binding of lactoferrin to

salivary agglutinin (B). A, After bovine lactoferrin (50 µg/ml) was treated at 40 to 100°C for 15 min,

the samples were subjected to surface plasmon resonance analysis. B, Reactions were carried out with

salivary lactoferrin (50 µg/ml) in 10 mM potassium phosphate buffer (pH 2 to 8) containing 0.15 M

NaCl. The binding of lactoferrin to salivary agglutinin is expressed as RU determined by surface

plasmon resonance. Values are given as the means ± S.D. of triplicate assays.

FIG. 5. Inhibition of saliva-induced aggregation of S. mutans cells by lactoferrin fragments. S.

mutans MT8148 cells grown in BHI broth were harvested and resuspended in aggregation buffer. The

suspensions were adjusted to an A550 of approximately 1.5 with aggregation buffer. The cell suspensions

(1 ml) were mixed with 25 µl of whole saliva and 1 nM of lactoferrin or lactoferrin fragment, and the

total volume of the reaction mixture was adjusted to 1.5 ml. Aggregation was measured by the reduction

in A550 after 2 h. Percent inhibition was calculated as 100 × [(a - b)/a], where a is the mean value without

lactoferrin preparation (control), and b is the mean value with lactoferrin preparation. Values are given as

the means ± S.D. of triplicate assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (compared with DHFR).


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TABLE I

Effects of various milk components on the saliva-induced aggregation of

S. mutans MT8148 cells a

_____________________________________________________________________

Milk component Aggregationb

% Inhibitionc

_____________________________________________________________________

Control 0.60 ± 0.10

α-Casein 0.51 ± 0.11 15.0

β-Casein 0.56 ± 0.15 6.7

γ-Casein 0.59 ± 0.17 1.7

κ-Casein 0.52 ± 0.18 13.3

Immunoglobulin G 0.56 ± 0.12 6.7

Lactalbumin 0.58 ± 0.14 3.3

Lactoferrin 0.14 ± 0.03d

76.7

Lactoglobulin 0.55 ± 0.17 8.3

Lactoperoxidase 0.44 ± 0.19 26.7

_____________________________________________________________________

a S. mutans MT8148 cells grown in BHI broth were harvested and resuspended in aggregation buffer.

The suspensions were adjusted to an A550 of approximately 1.5 with aggregation buffer. The cell

suspensions (1 ml) were mixed with 25 µl of whole saliva, 1 nM of each milk component, and the total

volume of the reaction mixture was adjusted to 1.5 ml.

b

Expressed as the reduction of A550 after 2 h. Values are the means ± S.D. of triplicate assays.


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c

Percent inhibition was calculated as 100 × [(a - b)/a], where a is the mean value without inhibitor

(control), and b is the mean value with inhibitor.

d p < 0.01 compared with control.


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TABLE II

Effect of lactoferrin on the saliva-induced aggregation of

streptococcal cells a

_____________________________________________________________________

Aggregationb

Strain ________________________ % Inhibitionc

Control Lactoferrin

_____________________________________________________________________

S. mutans

MT8148 0.60 ± 0.10 0.14 ± 0.03 77.3

Xc 0.63 ± 0.17 0.34 ± 0.10 51.0

S. sanguinis

ATCC 10556 0.26 ± 0.06 0.12 ± 0.02 53.9

S. oralis

ATCC 10557 0.67 ± 0.12 0.25 ± 0.03 62.7

S. gordonii

ATCC 10558 0.73 ± 0.04 0.24 ± 0.08 66.5

_____________________________________________________________________

a Streptococcal cells grown in BHI broth were harvested and resuspended in aggregation buffer. The

suspensions were adjusted to an A550 of approximately 1.5 with aggregation buffer. The cell suspensions

(1 ml) were mixed with 25 µl of whole saliva in the absence (control), or presence of 50 µg of lactoferrin,

and the total volume of the reaction mixture was adjusted to 1.5 ml.

b Expressed as the reduction of A550 after 2 h. Values are the means ± S.D. of triplicate assays.

c Percent inhibition was calculated as 100 I [(a - b)/a], where a is the mean value without lactoferrin

(control), and b is the mean value with lactoferrin.


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Morihide Mitoma, Takahiko Oho, Yoshihiro Shimazaki and Toshihiko Kogastreptococcal surface protein antigen and human salivary agglutinin

Inhibitory effect of bovine milk lactoferrin on the interaction between a

published online March 13, 2001J. Biol. Chem.

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