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Inhibitory Effect of Bovine Milk Lactoferrin on the Interaction between a Streptococcal Surface Protein Antigen and Human Salivary Agglutinin

2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês

10.1074/jbc.m101459200

ISSN

1083-351X

Autores

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

Tópico(s)

Pediatric Hepatobiliary Diseases and Treatments

Resumo

Human whole saliva induces aggregation ofStreptococcus 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 473–538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473–538 of bovine lactoferrin are important in the inhibition of saliva-induced aggregation of S. mutans. Human whole saliva induces aggregation ofStreptococcus 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 473–538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473–538 of bovine lactoferrin are important in the inhibition of saliva-induced aggregation of S. mutans. protein antigen serotype c recombinant PAc secretory immunoglobulin A brain heart infusion fast protein liquid chromatography resonance unit(s) dihydrofolate reductase polyacrylamide gel electrophoresis Streptococcus mutans has been strongly implicated in causation of dental caries, a common human disease (1Russell M.W. Curr. Opin. Dent. 1992; 2: 72-80PubMed Google Scholar, 2Russell R.R.B. Caries Res. 1994; 28: 69-82Crossref PubMed Scopus (62) Google Scholar). Colonization of the tooth surface by S. mutans is initiated by binding of the organism to salivary components on tooth surfaces (3Koga T. Okahashi N. Takahashi I. Kanamoto T. Asakawa H. Iwaki M. Infect. Immun. 1990; 58: 289-296Crossref PubMed Google Scholar). This binding is mediated by a 190-kDa surface protein antigen (PAc)1 of S. mutans, variously designated as antigen I/II, B, IF, P1, SR, and MSL-1 (1Russell M.W. Curr. Opin. Dent. 1992; 2: 72-80PubMed Google Scholar, 3Koga T. Okahashi N. Takahashi I. Kanamoto T. Asakawa H. Iwaki M. Infect. Immun. 1990; 58: 289-296Crossref PubMed Google Scholar, 4Bowen W.H. Schilling K. Giertsen E. Pearson S. Lee S.F. Bleiweis A. Beeman D. Infect. Immun. 1991; 59: 4606-4609Crossref PubMed Google Scholar, 5Lee S.F. Progulske-Fox A. Erdos G.W. Piacentini D.A. Ayakawa G.Y. Crowley P.J. Bleiweis A.S. Infect. Immun. 1989; 57: 3306-3313Crossref PubMed Google Scholar). Various salivary components have been reported to bind to S. mutans or to induce its aggregation (6Payne J.B. Iacono V.J. Crawford I.T. Lepre B.M. Bernzweig E. Grossbard B.L. Oral Microbiol. Immunol. 1991; 6: 169-176Crossref PubMed Scopus (23) Google Scholar, 7Carlén A. Olsson J. J. Dent. Res. 1995; 74: 1040-1047Crossref PubMed Scopus (34) Google Scholar, 8Senpuku H. Kato H. Todoroki M. Hanada N. Nishizawa T. FEMS Microbiol. Lett. 1996; 139: 195-201Crossref PubMed Google Scholar). 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) (9Oho T., Yu, H. Yamashita Y. Koga T. Infect. Immun. 1998; 66: 115-121Crossref PubMed Google Scholar). 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 (10Ebina T. Ohta M. Kanamura Y. Ymamoto-Osumi Y. Baba K. J. Med. Virol. 1992; 38: 117-123Crossref PubMed Scopus (59) Google Scholar, 11Ishida A. Yoshikai Y. Murosaki S. Kubo C. Hidaka Y. Nomoto K. J. Nutr. 1992; 122: 1875-1883Crossref PubMed Scopus (9) Google Scholar, 12Murosaki S. Yoshikai Y. Kubo C. Ishida A. Matsuzaki G. Sato T. Endo K. Nomoto K. J. Nutr. 1991; 121: 1860-1868Crossref PubMed Scopus (12) Google Scholar, 13Freedman D.J. Tacket C.O. Delehanty A. Maneval D.R. Nataro J. Crabb J.H. J. Infect. Dis. 1998; 177: 662-667Crossref PubMed Scopus (113) Google Scholar). Bovine milk contains several protein components, including caseins, immunoglobulins, lactalbumin, lactoferrin, lactoglobulin, lactoperoxidase, and lysozyme (14Mulvihill D.M. Grufferty M.B. Adv. Exp. Med. Biol. 1997; 415: 77-93Crossref PubMed Scopus (4) Google Scholar). Casein and lactoperoxidase have been reported to inhibit the adherence of S. mutans to saliva-coated hydroxyapatite (15Roger V. Tenovuo J. Lenander-Lumikari M. Söderling E. Vilja P. Caries Res. 1994; 28: 421-428Crossref PubMed Scopus (45) Google Scholar,16Vacca-Smith A.M. van Wuyckhuyse B.C. Tabak L.A. Bowen W.H. Arch. Oral Biol. 1994; 39: 1063-1069Crossref PubMed Scopus (81) Google Scholar). κ-Casein reduces the glucosyltransferase activity of S. mutans, which in turn reduces glucan formation (17Vacca-Smith A.M. Bowen W.H. Caries Res. 1995; 29: 498-506Crossref PubMed Scopus (27) Google Scholar), and lactoferrin has a bactericidal effect on S. mutans (18Lassiter M.O. Newsome A.L. Sams L.D. Arnold R.R. J. Dent. Res. 1987; 66: 480-485Crossref PubMed Scopus (52) Google Scholar). 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. S. mutans strains MT8148 (3Koga T. Okahashi N. Takahashi I. Kanamoto T. Asakawa H. Iwaki M. Infect. Immun. 1990; 58: 289-296Crossref PubMed Google Scholar) and Xc (19Tsukioka Y. Yamashita Y. Nakano Y. Oho T. Koga T. J. Bacteriol. 1997; 179: 4411-4414Crossref PubMed Google Scholar) were used as representative strains of S. mutansserotype c. S. mutans TK18 is a recombinant strain that produces a large amount of PAc (3Koga T. Okahashi N. Takahashi I. Kanamoto T. Asakawa H. Iwaki M. Infect. Immun. 1990; 58: 289-296Crossref PubMed Google Scholar). Streptococcus sanguinis ATCC 10556, Streptococcus oralisATCC 10557, and Streptococcus gordonii ATCC 10558 were used as type strains. Escherichia coli M15[pREP4] was obtained from Qiagen. The culture media used were 2 × TY broth (20Laloi P. Munro C.L. Jones K.R. Macrina F.L. Infect. Immun. 1996; 64: 28-36Crossref PubMed Google Scholar) for Escherichia coli and brain heart infusion (BHI, Difco) broth for streptococci. 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 was isolated by the method of Oho et al. (9Oho T., Yu, H. Yamashita Y. Koga T. Infect. Immun. 1998; 66: 115-121Crossref PubMed Google Scholar). Briefly, clarified whole saliva diluted 1/2 with aggregation buffer (1.5 mmKH2PO4 (pH 7.2), 6.5 mmNa2HPO4, 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 a 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 (9Oho T., Yu, H. Yamashita Y. Koga T. Infect. Immun. 1998; 66: 115-121Crossref PubMed Google Scholar). 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) (3Koga T. Okahashi N. Takahashi I. Kanamoto T. Asakawa H. Iwaki M. Infect. Immun. 1990; 58: 289-296Crossref PubMed Google Scholar). 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. (21Oho T. Shimazaki Y. Mitoma M. Yoshimura M. Yamashita Y. Okano K. Nakano Y. Kawagoe H. Fukuyama M. Fujihara N. Koga T. J. Nutr. 1999; 129: 1836-1841Crossref PubMed Scopus (24) Google Scholar). Iron-saturated bovine lactoferrin and iron-free lactoferrin (apolactoferrin) were prepared from bovine lactoferrin according to the methods of Kawasaki et al. (22Kawasaki Y. Tazume S. Shimizu K. Matsuzawa H. Dosako S. Isoda H. Tsukiji M. Fujimura R. Muranaka Y. Ishida H. Biosci. Biotechnol. Biochem. 2000; 64: 348-354Crossref PubMed Scopus (52) Google Scholar) and Shimazaki et al. (23Shimazaki K. Kawano N. Yoo Y.C. Comp. Biochem. Physiol. 1991; 98: 417-422Crossref Scopus (10) Google Scholar), 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., Zama, Japan. Protein content was determined according to the method of Lowry et al. (24Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), with bovine serum albumin as a standard. 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 mmimidazole 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. 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 (Shimadzu, Kyoto, Japan). Streptococcal cells were suspended in aggregation buffer at an A550 of ∼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 UV-visible recording spectrophotometer (Ultrospec 3000, Amersham Pharmacia Biotech). Surface plasmon resonance, which permits real-time analysis of macromolecular interactions (25Jönsson U. Fägerstam L. Ivarsson B. Johnsson B. Karlsson R. Lundh K. Löfås S. Persson B. Roos H. Rönnberg I. Sjölander S. Stenberg E. Ståhlberg R. Urbaniczky C. Östlin H. Malmqvist M. BioTechniques. 1991; 11: 620-627PubMed Google Scholar), 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 Johnssonet al. (26Johnsson B. Löfås S. Lindquist G. Anal. Biochem. 1991; 198: 268-277Crossref PubMed Scopus (1202) Google Scholar). 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 phosphate-buffered saline (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, 7.4, 7.1, and 10.9 ng/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 2–8) containing 0.15 m NaCl. Divalent cation specificity was examined in phosphate-buffered saline (pH 7.0) containing 0–2 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 ∼1 ng/mm2 (27Stenberg E. Persson B. Roos H. Urbaniczky C. J. Colloid Interface Sci. 1991; 143: 513-526Crossref Scopus (1005) Google Scholar). In thermal stability studies, lactoferrin was heated at 40–100 °C for 15 min and was then subjected to the surface plasmon resonance binding assay. Truncated bovine lactoferrin fragments were prepared as 6 × His-tagged fusion proteins by cloning of polymerase chain reaction-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′-TATAGGATCCCTGATCGTCAACCAGACA-3′. The amplified DNAs were digested with either BamHI and SalI orSacI and SalI (LfN only) restriction sites (underlined) and inserted into the BamHI-SalI orSacI-SalI sites of the pQE-30 plasmid. The ligated DNAs were then transformed into E. coliM15[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 a DNA fragment encoding the DHFR, was transformed into E. coliM15[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, 6m 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 theA280 of eluate was less than 0.01. The fusion proteins were eluted with buffer B containing 250 mmimidazole. The eluted proteins were refolded by sequential dialysis against buffers containing decreasing urea concentrations for 18 h in each buffer at 4 °C (28Wingfield P.T. Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. 1. John Wiley and Sons, New York1995: 6.1.1-6.2.15Google Scholar). The gradient buffers contained 4, 2, and 1m urea in 0.1 m Tris-HCl (pH 8.0), 0.1m 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 was performed using 12.5 and 15% polyacrylamide gels according to the method of Laemmli (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar). 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 (30Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5906) Google Scholar). 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). Differences between the control and the test samples in aggregation were determined by Student'st test. The FPLC fraction of bovine milk eluted at 0.64m 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. 2 A, lane 2). In Western blot, rabbit anti-bovine lactoferrin antiserum reacted with this band (Fig. 2 B, 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 (31Bellamy W. Takase M. Yamauchi K. Wakabayashi H. Kawase K. Tomita M. Biochim. Biophys. Acta. 1992; 1121: 130-136Crossref PubMed Scopus (814) Google Scholar). These results indicated that the aggregation inhibitory component is lactoferrin.Figure 2SDS-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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Aggregation of the typicalS. mutans strain MT8148 (serotype c) in the presence of whole saliva or salivary agglutinin was examined by a spectrophotometric 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 (TableI). 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 (TableII).Table IEffects of various milk components on the saliva-induced aggregation of S. mutans MT8148 cellsMilk componentAggregation1-aExpressed as the reduction ofA550 after 2 h. Values are the means ± S.D. of triplicate assays.InhibitionbPercent inhibition was calculated as 100 × [(a − b)/a], where a is the mean value without inhibitor (control), andb is the mean value with inhibitor.%Control0.60 ± 0.10α-Casein0.51 ± 0.1115.0β-Casein0.56 ± 0.156.7γ-Casein0.59 ± 0.171.7κ-Casein0.52 ± 0.1813.3Immunoglobulin G0.56 ± 0.126.7Lactalbumin0.58 ± 0.143.3Lactoferrin0.14 ± 0.03cp < 0.01 compared with control.76.7Lactoglobulin0.55 ± 0.178.3Lactoperoxidase0.44 ± 0.1926.7S. mutans MT8148 cells grown in BHI broth were harvested and resuspended in aggregation buffer. The suspensions were adjusted to anA550 of approximately 1.5 with aggregation buffer. The cell suspensions (1 ml) were mixed with 25 μl of whole saliva, 1 nm milk component, and the total volume of the reaction mixture was adjusted to 1.5 ml.1-a Expressed as the reduction ofA550 after 2 h. Values are the means ± S.D. of triplicate assays.1-b Percent inhibition was calculated as 100 × [(a − b)/a], where a is the mean value without inhibitor (control), andb is the mean value with inhibitor.1-c p < 0.01 compared with control. Open table in a new tab Table IIEffect of lactoferrin on the saliva-induced aggregation of streptococcal cellsStrainAggregation 2-aExpressed as the reduction ofA550 after 2 h. Values are the means ± S.D. of triplicate assays.Inhibition2-bPercent inhibition was calculated as 100 × [(a − b/a], where a is the mean value without lactoferrin (control), andb is the mean value with lactoferrin.ControlLactoferrin%S. mutansMT81480.60 ± 0.100.14 ± 0.0377.3Xc0.63 ± 0.170.34 ± 0.1051.0S. sanguinisATCC 105560.26 ± 0.060.12 ± 0.0253.9S. oralisATCC 105570.67 ± 0.120.25 ± 0.0362.7S. gordoniiATCC 105580.73 ± 0.040.24 ± 0.0866.5Streptococcal cells grown in BHI broth were harvested and resuspended in aggregation buffer. The suspensions were adjusted to anA550 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.a Expressed as the reduction ofA550 after 2 h. Values are the means ± S.D. of triplicate assays.2-b Percent inhibition was calculated as 100 × [(a − b/a], where a is the mean value without lactoferrin (control), andb is the mean value with lactoferrin. Open table in a new tab S. mutans MT8148 cells grown in BHI broth were harvested and resuspended in aggregation buffer. The suspensions were adjusted to anA550 of approximately 1.5 with aggregation buffer. The cell suspensions (1 ml) were mixed with 25 μl of whole saliva, 1 nm milk component, and the total volume of the reaction mixture was adjusted to 1.5 ml. Streptococcal cells grown in BHI broth were harvested and resuspended in aggregation buffer. The suspensions were adjusted to anA550 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. 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 phosphate-buffered saline (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.4 A). 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. 4 B). 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 a single band (data not shown). The N-terminally truncated lactoferrin fragment, LfC (residues 345–689), strongly inhibited saliva-induced aggregation ofS. mutans cells, whereas the C-terminally truncated fragment LfN (residues 1–344) weakly inhibited the aggregation (Fig.5). Fragments Lf4 (residues 345–571), Lf41 (residues 345–538), Lf45 (residues 366–571), Lf46 (residues 399–571), and Lf47 (residues 432–571) also exhibited strong inhibition of saliva-induced aggregation of S. mutans, as did the shorter fragment Lf411 (residues 473–538). In contrast, fragments Lf43 (residues 345–472) and Lf44 (residues 345–439) exhibited only weak inhibitory activity. The 6 × His-tagged DHFR, which was used as control, also weakly inhibited aggregation. 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 (3Koga T. Okahashi N. Takahashi I. Kanamoto T. Asakawa H. Iwaki M. Infect. Immun. 1990; 58: 289-296Crossref PubMed Google Scholar, 9Oho T., Yu, H. Yamashita Y. Koga T. Infect. Immun. 1998; 66: 115-121Crossref PubMed Google Scholar). Gonget al. (32Gong K. Mailoux L. Herzberg M.C. J. Biol. Chem. 2000; 275: 8970-8974Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) 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 ∼8.0 (33Shimazaki K. Kawaguchi A. Sato T. Ueda Y. Tomimura T. Shimamura S. Int. J. Biochem. 1993; 25: 1653-1658Crossref PubMed Scopus (27) Google Scholar). 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 (34Saito H. Takase M. Tamura Y. Shimamura S. Tomita M. Adv. Exp. Med. Biol. 1994; 357: 219-226Crossref PubMed Scopus (18) Google Scholar). 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 (35Baker E.N. Anderson B.F. Baker H.M. MacGillivray R.T.A. Moore S.A. Peterson N.A. Shewry S.C. Tweedie J.W. Adv. Exp. Med. Biol. 1998; 443: 1-14Crossref PubMed Scopus (47) Google Scholar, 36Sánchez L. Caivo M. Brock J.H. Arch. Dis. Child. 1992; 67: 657-661Crossref PubMed Scopus (382) Google Scholar). The lactoferrin preparation used in this study was 19.3% iron-saturated. To examine the role of iron binding in inhibition of S. mutans aggregation, we also prepared apolactoferrin 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. (37Soukka T. Roger V. Söderling E. Tenovuo J. Microb. Ecol. Health Dis. 1994; 7: 139-144Google Scholar), who observed that these three types of lactoferrin cause no difference in the binding of S. mutans, although 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.(38Soukka T. Tenovuo J. Rundegren J. Arch. Oral Biol. 1993; 38: 227-232Crossref PubMed Scopus (23) Google Scholar) showed that apolactoferrin effectively agglutinates S. mutans cells but not the other bacteria. However, our preliminary studies have shown that all of the three types of lactoferrin did not induce the aggregation of S. mutanscells. 2M. Mitoma, T. Oho, Y. Shimazaki, and T. Koga, unpublished data. 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. mutanscells. Our results suggest that lactoferrin residues 473–538 play an important role in the inhibition of saliva-induced aggregation ofS. mutans. Other fragments lacking these residues, such as LfN (residues 1–344), Lf43 (residues 345–472), and Lf44 (residues 345–439), exhibited only weak inhibitory activity. The lactoferrin molecule is proposed to consist of two lobes (N-lobe and C-lobe) (40Moore S.A. Anderson B.F. Groom C.R. Haridas M. Baker E.N. J. Mol. Biol. 1997; 274: 222-236Crossref PubMed Scopus (336) Google Scholar). The N-lobe contains the active domains for bactericidal action and heparin binding (31Bellamy W. Takase M. Yamauchi K. Wakabayashi H. Kawase K. Tomita M. Biochim. Biophys. Acta. 1992; 1121: 130-136Crossref PubMed Scopus (814) Google Scholar, 41Shimazaki K. Uji K. Tazume T. Kumura H. Shimo-Oka T. Shimazaki K. Tsuda H. Tomita M. Kuwata T. Perraudin J.-P. Lactoferrin: Structure, Function and Applications. Elsevier Science Publishers B. V., Amsterdam, The Netherlands2000: 37-46Google Scholar), whereas the C-lobe contains a functional domain for hepatocyte binding and internalization (42Maheshwari P. Sitaram P. Mcabee D.D. Biochem. J. 1997; 323: 815-822Crossref PubMed Scopus (15) Google Scholar). 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 (43Chou P.Y. Fasman G.D. Biochemistry. 1974; 13: 222-245Crossref PubMed Scopus (2541) Google Scholar). Although all the active fragments containing residues 473–538 had acidic pI values, the inactive fragment Lf44 also had an acidic pI value (pI = 5.2). 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 (44Yamauchi K. Tomita M. Giehl T.J. Ellison III, R.T. Infect. Immun. 1993; 61: 719-728Crossref PubMed Google Scholar). 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 (45Baker H.M. Anderson B.F. Kidd R.D. Shewry S.C. Baker E.N. Shimazaki K. Tsuda H. Tomita M. Kuwata T. Perraudin J.-P. Lactoferrin: Structure, Function and Applications. Elsevier Science Publishers B. V., Amsterdam, The Netherlands2000: 3-15Google Scholar). 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 ofS. 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 (46Gibbons R.J. Hay D.I. Infect. Immun. 1988; 56: 439-445Crossref PubMed Google Scholar) and Raj et al. (47Raj P.A. Johnsson M. Levine M.J. Nancollas G.H. J. Biol. Chem. 1992; 267: 5968-5976Abstract Full Text PDF PubMed Google Scholar) 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 (48Gibbons R.J. J. Dent. Res. 1989; 68: 750-760Crossref PubMed Scopus (333) Google Scholar) proposed a model that an apparent conformational change occurs when salivary components bind to hydroxyapatite, 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 (39Brock J.H. Hutchens T.W. Lönnerdal B. Lactoferrin: Interactions and Biological Functions. Humana Press, Totowa, NJ1997: 3-23Crossref Google Scholar). 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 (18Lassiter M.O. Newsome A.L. Sams L.D. Arnold R.R. J. Dent. Res. 1987; 66: 480-485Crossref PubMed Scopus (52) Google Scholar). 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 473–538 of bovine lactoferrin play an important role in the interaction of lactoferrin with salivary agglutinin. 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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