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Inhibitory Effect of Bovine Milk Lactoferrin on the Interaction between a Streptococcal Surface Protein Antigen and Human Salivary Agglutinin*
Received for publication,February 15,2001,and in revised form,March 6,2001Published,JBC Papers in Press,March 13,2001,DOI 10.1074/jbc.M101459200
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
Human whole saliva induces aggregation of Strepto-coccus mutans cells via an interaction between a surface protein antigen (PAc)of the organism and salivary ag-glutinin.Bovine milk inhibits the saliva-induced aggre-gation of S.mutans .In this study,the milk component that possesses inhibitory activity against this aggrega-tion was isolated and found to be lactoferrin.Surface plasmon resonance analysis indicated that bovine lacto-ferrin binds more strongly to salivary agglutinin,espe-cially 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 lacto-ferrin,11truncated bovine lactoferrin fragments were constructed.A fragment corresponding to the C-termi-nal half of the lactoferrin molecule had a strong inhibi-tory effect on the saliva-induced aggregation of S.mu-tans ,whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect.Seven shorter fragments corresponding to lactoferrin residues 473–538also showed a high ability to inhibit the aggre-gation of S.mutans .These results suggest that residues 473–538of bovine lactoferrin are important in the inhi-bition of saliva-induced aggregation of S.mutans .
Streptococcus mutans has been strongly implicated in causa-tion of dental caries,a common human disease (1,2).Coloni-zation 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 (PAc)1of S.mutans ,variously designated as antigen I/II,B,IF,P1,SR,and 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 patho-gens (10–13).Bovine milk contains several protein compo-nents,including caseins,immunoglobulins,lactalbumin,lacto-ferrin,lactoglobulin,lactoperoxidase,and lysozyme (14).Casein and lactoperoxidase have been reported to inhibit the adherence of S.mutans to saliva-coated hydroxyapatite (15,16).␬-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,dele-tion 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 TK18is a recombinant strain that produces a large amount of PAc (3).Streptococcus sanguinis ATCC 10556,Streptococcus oralis ATCC 10557,and Streptococcus gordonii ATCC 10558were used as type strains.Escherichia coli M15[pREP4]was obtained from Qiagen.The culture media used were 2ϫTY broth (20)for Escherichia coli and brain heart infusion (BHI,Difco)broth for streptococci.
Saliva—Unstimulated whole saliva was collected from a single donor (male,42years of age)in an ice-chilled tube and clarified by centrifu-gation at 12,000ϫg for 10min.
Salivary Agglutinin—Salivary agglutinin was isolated by the method of Oho et al.(9).Briefly,clarified whole saliva diluted 1/2with aggre-gation buffer (1.5m M KH 2PO 4(pH 7.2),6.5m M Na 2HPO 4,2.7m M KCl,137m M NaCl)was incubated with an equal volume of a cell suspension of S.mutans MT8148at 37°C for 30min.Cells were collected by centrifugation and washed with aggregation buffer,and adsorbed sal-ivary agglutinin was eluted with the same buffer supplemented with 1m M EDTA.The eluate was filtered (0.2-␮m pore size)and subjected to gel filtration chromatography on a Superdex 200HR (Amersham Phar-macia 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).
Recombinant PAc (rPAc)—rPAc was purified from the culture super-natants of transformant S.mutans TK18by ammonium sulfate precip-itation,chromatography on DEAE-cellulose,and subsequent gel filtra-tion on Sepharose CL-6B (Amersham Pharmacia Biotech)(3).
Milk Components—Bovine ␣-casein,␤-casein,␬-casein,lactalbumin,lactoferrin,and lactoperoxidase were purchased from Sigma.Bovine ␥-casein was purchased from Research Organics,and bovine lactoglob-ulin from ICN Biomedicals.Bovine immunoglobulin G was prepared from bovine milk,using affinity chromatography on a HiTrap protein G column (5ml)(Amersham Pharmacia Biotech)according to the method
*This work was supported in part by Grants-in-aid for Developmen-tal Scientific Research (A)12357013(to T.K.)and (C)11672051(to 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 (to T.K.).The costs of publica-tion of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.‡To whom correspondence should be addressed.Tel.:81-92-642-6353;Fax:81-92-642-6354;E-mail:oho@dent.kyushu-u.ac.jp.1
The abbreviations used are:PAc,protein antigen serotype c;rPAc,recombinant PAc;sIgA,secretory immunoglobulin A;BHI,brain heart infusion;FPLC,fast protein liquid chromatography;RU,resonance unit(s);DHFR,dihydrofolate reductase;PAGE,polyacrylamide gel electrophoresis.
T HE J OURNAL OF B IOLOGICAL C HEMISTRY
Vol.276,No.21,Issue of May 25,pp.18060–18065,2001
©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.
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of Oho et al.(21).Iron-saturated bovine lactoferrin and iron-free lacto-ferrin(apolactoferrin)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 deter-mined by the Wako Fe-B test(Wako,Osaka,Japan).Bovine lactoferrin (Sigma)was determined to be19.3%ctoferricin B was a gift from the Nutrition Science Laboratory,Morinaga Milk In-dustry Co.,Zama,Japan.Protein content was determined according to the method of Lowry et al.(24),with bovine serum albumin as a standard.
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 at12,000ϫg for15min,and the skimmed milk was dialyzed against10m M imidazole HCl buffer(pH7.0).Then,the milk sample was passed through a0.2-␮m filter and applied to a Mono S HR 5/5column(Amersham Pharmacia Biotech)that had been equilibrated with10m M imidazole HCl buffer(pH7.0).After sample application,the column was washed with5volumes of the same buffer,and the bound material was eluted with a linear gradient(0–1M)of NaCl in the same buffer.Each fraction was analyzed for protein by monitoring the ab-
sorbance at280nm(A
280)and was assayed for aggregation inhibitory
activity.
Sequence Determination—The N-terminal amino acid sequence of the isolated aggregation inhibitory bovine milk component was deter-mined by Edman degradation using a Shimadzu PSSQ-21gas-phase sequencer(Shimadzu,Kyoto,Japan).
Aggregation Assay—Streptococcal cells were suspended in aggrega-
tion buffer at an A
550ofϳ1.5.Either25␮l of whole saliva or10␮l of
salivary agglutinin(0.5mg/ml)was mixed with1ml of the cell suspen-sion and various amounts of bovine milk component,and the total volume of the reaction mixture was adjusted to1.5ml with aggregation
buffer.CaCl
2was added to the mixture of salivary agglutinin at a final
concentration of1m M.Bacterial aggregation was determined by mon-
itoring the change in A
550at37°C for2h with a UV-visible recording
spectrophotometer(Ultrospec3000,Amersham Pharmacia Biotech).
Binding of Bovine Lactoferrin to rPAc or Salivary Agglutinin—Sur-face plasmon resonance,which permits real-time analysis of macromo-lecular 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 BIAcore2000sur-face plasmon resonance biosensor(Amersham Pharmacia Biotech). First,rPAc,salivary agglutinin,high molecular mass glycoprotein sep-arated by electrophoretic fractionation,and sIgA separated by electro-phoretic fractionation were immobilized on carboxymethylated,dext-ran-coated,gold-surfaced CM5sensor chips via primary amino group linkages according to the method of Johnsson et al.(26).For immobili-zation of each protein,35␮l of a300␮g/ml solution in10m M sodium acetate buffer(pH4.5)was passed over the activated chip surface,while phosphate-buffered saline(pH7.0)was maintained at5␮l/min throughout the immobilizing process.Binding of rPAc,salivary agglu-tinin,high molecular mass glycoprotein,and sIgA to the chip surfaces occurred at5.8,7.4,7.1,and10.9ng/mm2,respectively.Each milk component,diluted in an appropriate running buffer,was then passed over the immobilized surface at a flow rate of10␮l/min.The effect of pH on the binding of bovine lactoferrin to salivary agglutinin was assayed in10m M potassium phosphate buffer(pH2–8)containing0.15M NaCl. Divalent cation specificity was examined in phosphate-buffered saline
(pH7.0)containing0–2m M CaCl
2,MgCl
2
,or MnCl
2
.The dissociation
phase of binding was initiated by the injection of the diluent buffer at10␮l/min.All binding experiments were performed at25°C.The surface resonance signal in each binding cycle was expressed in resonance units (RU).A resonance of1,000RU corresponds to a shift of0.1°in the resonance angle,which corresponds to a change in surface protein concentration ofϳ1ng/mm2(27).
Heat Treatment—In thermal stability studies,lactoferrin was heated at40–100°C for15min and was then subjected to the surface plasmon resonance binding assay.
Bovine Lactoferrin Fragments—Truncated bovine lactoferrin frag-ments were prepared as6ϫHis-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,5Ј-TATAGAGCTCATGAAGCTCTTCGT-CCCC-3Ј;LfN-R,5Ј-ACACGTCGACTTACCTGGTGTACCGCGCCTT-3Ј;LfC-F,5Ј-TATAGGATCCGTCGTGTGGTGTGCCGTG-3Ј;LfC-R,5Ј-ACACGTCGACTTACCTCGTCAGGAAGGCGCA-3Ј;Lf4-R,5Ј-ACACG-TCGACTTACAACCTGAAGTCCTCACG-3Ј;Lf41-R,5Ј-ACACGTCGA-CTTACCCAACGTCCTCAGCCAG-3Ј;Lf42-R,5Ј-ACACGTCGACTTA-ACACAAGGCACAGAGTCT-3Ј;Lf43-R,5Ј-ACACGTCGACTTAGCCC-
ATGGGGATGTTCCA-3Ј;Lf44-R,5Ј-ACACGTCGACTTAGACAACTG-CCACGGCAAG-3Ј;Lf45-F,5Ј-TATAGGATCCGGCCAGAACGTGACC-TGT-3Ј;Lf46-F,5Ј-TATAGGATCCATCTACACTGCGGGCAAG-3Ј; Lf47-F,5Ј-TATAGGATCCGGGTACCTTGCCGTGGCA-3Ј;Lf411-F,5Ј-TATAGGATCCCTGATCGTCAACCAGACA-3Ј.The amplified DNAs were digested with either Bam HI and Sal I or Sac I and Sal I(LfN only) restriction sites(underlined)and inserted into the Bam HI-Sal I or Sac-I-Sal I sites of the pQE-30plasmid.The ligated DNAs were then tra-nsformed 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,6ϫHis-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.coli M15[pREP4].
Lactoferrin and DHFR fusion proteins were extracted from whole cell extracts of E.coli M15[pREP4]cells containing the recombinant plas-mids.Cells were cultured in2ϫTY broth containing100␮g/ml ampi-cillin and25␮g/ml kanamycin at37°C until an A
550
of1.0was attained. Expression was induced by addition of isopropyl-␤-D-thiogalactopyrano-side to the cultures at a final concentration of1m M,and the cultures were grown for3h.Cells were harvested by centrifugation at5,000ϫg for20min,and one-step purification of the fusion proteins was performed with Ni2ϩ-HiTrap chelating columns(1ml)(Amersham Pharmacia Biotech)according to the manufacture’s instructions.In brief,the cell pellet was solubilized in10m M Tris-HCl(pH8.0),0.1M sodium phosphate,6M guanidine HCl(buffer A)at5ml/g and mixed by inversion for1h at4°C.The lysate was centrifuged at10,000ϫg for 20min at4°C,and the cleared supernatant was applied to a Ni2ϩ-HiTrap chelating column that had been equilibrated with buffer A.The column was extensively washed with buffer A and then with5or more volumes of10m M Tris-HCl(pH8.0),0.1M sodium phosphate,8M urea
(buffer B)containing10m M imidazole until the A
280
of eluate was less than0.01.The fusion proteins were eluted with buffer B containing250 m M imidazole.
The eluted proteins were refolded by sequential dialysis against buffers containing decreasing urea concentrations for18h in each buffer at4°C(28).The gradient buffers contained4,2,and1M urea in 0.1M Tris-HCl(pH8.0),0.1M sodium phosphate,and2m M dithiothre-itol.After dialysis against1M urea,fusion proteins were dialyzed against50m M sodium phosphate(pH8.0)containing0.3M NaCl for 18h at4°C.Each fusion protein was analyzed by SDS-PAGE.
SDS-PAGE and Western Blotting—SDS-PAGE was performed using 12.5and15%polyacrylamide gels according to the method of Laemmli (29).After electrophoresis,the gels were stained with Coomassie Bril-liant Blue R-250.Electrophoresis calibration kits(Amersham Pharma-cia Biotech)were used as molecular mass markers.For Western blot-ting,samples were subjected to SDS-PAGE and transferred electrophoretically to nitrocellulose membranes according to the method of Burnette(30).After blocking with1%bovine serum albumin in Tris-buffered saline(20m M Tris-HCl(pH7.5),150m M NaCl)con-taining1%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 at0.64M NaCl inhibited the saliva-induced aggregation of S. mutans cells(Fig.1).Coomassie staining of the SDS gel re-vealed a single80-kDa band in this fraction(Fig.2A,lane2).In Western blot,rabbit anti-bovine lactoferrin antiserum reacted with this band(Fig.2B,lane1).The N-terminal amino acid sequence of this component was Ala-Pro-Arg-Lys-Asn-Val-Arg-
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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 typi-cal S.mutans strain MT8148(serotype c)in the presence of whole saliva or salivary agglutinin was examined by a spectro-photometric assay.Both whole saliva and salivary agglutinin induced strong aggregation.Testing of various bovine milk components revealed that lactoferrin inhibited this saliva-in-duced 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 lac-toperoxidase,␣-casein,and ␬-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.gor-donii ATCC 10558,were also tested for their ability to aggre-
gate in the presence of whole saliva with or without bovine lactoferrin.Bovine lactoferrin showed the same inhibitory ef-fect 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 Aggluti-nin—The binding of bovine lactoferrin to rPAc,salivary agglu-tinin,or to components of salivary agglutinin separated by electrophoretic fractionation was analyzed by surface plasmon ctoferrin (50␮g/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 149Ϯ16,470Ϯ13,718Ϯ47,and 34Ϯ1RU/ng of immobilized ligand,respectively (mean ϮS.D.of triplicate assays).
Binding of bovine lactoferrin to immobilized salivary agglu-tinin was enhanced by the addition of CaCl 2to the running buffer,with an optimum concentration of 0.5m M CaCl 2.MgCl
2
F I
G .1.Fractionation of bovine milk by FPLC.Ten ml of defatted milk were dialyzed against 10m M imidazole HCl buffer (p
H 7.0)and then applied to a Mono S HR 5/5column.The bound mate-rial was eluted with a linear gradient of NaCl (0–1.0M )in 10m M imidazole HCl buffer (pH 7.0).Fractions were monitored for protein by their absorbance at 280nm (q )and for their inhibitory effect on the aggregation of S.mutans cells (E ).—-—-—,NaCl
gradient.
F I
G .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-mercap-toethanol)and heated at 100°C for 3min.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 ␣-lactalbumin (14.4kDa),soybean trypsin inhibitor (20kDa),carbonic anhydrase (30kDa),ovalbumin (43kDa),bovine serum albu-min (67kDa),and phosphorylase b (94kDa).Lanes :1,defatted bovine milk (5␮g);2,the aggregation inhibitory protein (3␮g);3,bovine lactoferrin from Sigma (3␮g).B ,milk proteins on the gel were electro-phoretically transferred to a nitrocellulose membrane,and the mem-brane was reacted with goat antiserum against bovine nes :1,the aggregation inhibitory protein (2␮g);2,bovine lactoferrin from Sigma (2␮
g).
F I
G .3.Dose-dependent inhibition of the saliva-induced aggre-gation of S.mutans cells by bovine lactoferrin.S.mutans MT8148cells grown in BHI broth were harvested and resuspended in aggrega-tion buffer.The suspensions were adjusted to an A 550of ϳ1.5with aggregation buffer.The cell suspensions (1ml)were mixed with 25␮l of whole saliva and various amounts of lactoferrin,and the total volume of the reaction mixture was adjusted to 1.5ml.Aggregation was measured by the reduction in A 550after 2h.Percent inhibition was calculated as 100ϫ[(a Ϫb )/a ],where a is the mean value without lactoferrin (control),and b is the mean value with lactoferrin.Values are given as the means ϮS.D.of triplicate assays.
Binding of Bovine Lactoferrin to Salivary Agglutinin
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and MnCl 2did not enhance binding (data not shown).In ther-mal stability studies,the biosensor response induced by bind-ing of bovine lactoferrin to immobilized salivary agglutinin gradually decreased as the temperature used to heat the lacto-ferrin 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 aggluti-nin 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 bo-vine lactoferrin molecule,116ϫHis-tagged lactoferrin frag-ments were cloned and expressed in E.coli.These fusion pro-teins 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 trun-cated lactoferrin fragment,LfC (residues 345–689),strongly inhibited saliva-induced aggregation of S.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 aggrega-tion 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 activ-ity.The 6ϫHis-tagged DHFR,which was used as control,also weakly inhibited aggregation.
DISCUSSION
Human saliva induces aggregation of S.mutans via an in-teraction 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 ␣-amylase,which forms a S.sanguinis -binding site.In this study,we showed that bovine milk lacto-
T ABLE I
Effects of various milk components on the saliva-induced aggregation
of S.mutans MT8148cells
S.mutans MT8148cells grown in BHI broth were harvested and resuspended in aggregation buffer.The suspensions were adjusted to an A 550of approximately 1.5with aggregation buffer.The cell suspen-sions (1ml)were mixed with 25␮l of whole saliva,1n M milk compo-nent,and the total volume of the reaction mixture was adjusted to 1.5ml.
Milk component
Aggregation a
Inhibition b
%
Control 0.60Ϯ0.10␣-Casein 0.51Ϯ0.1115.0␤-Casein 0.56Ϯ0.15 6.7␥-Casein 0.59Ϯ0.17 1.7␬-Casein
0.52Ϯ0.1813.3Immunoglobulin G 0.56Ϯ0.12 6.7Lactalbumin 0.58Ϯ0.14 3.3Lactoferrin 0.14Ϯ0.03c 76.7Lactoglobulin 0.55Ϯ0.178.3Lactoperoxidase
0.44Ϯ0.19
26.7
a
Expressed as the reduction of A 550after 2h.Values are the means ϮS.D.of triplicate assays.b
Percent inhibition was calculated as 100ϫ[(a Ϫb )/a ],where a is the mean value without inhibitor (control),and b is the mean value with inhibitor.c
p Ͻ0.01compared with control.T ABLE II
Effect of lactoferrin on the saliva-induced aggregation
of streptococcal cells
Streptococcal cells grown in BHI broth were harvested and resus-pended in aggregation buffer.The suspensions were adjusted to an A 550of approximately 1.5with aggregation buffer.The cell suspensions (1ml)were mixed with 25␮l of whole saliva in the absence (control)or presence of 50␮g of lactoferrin,and the total volume of the reaction mixture was adjusted to 1.5ml.
Strain
Aggregation a
Inhibition b
Control
Lactoferrin
%
S.mutans MT81480.60Ϯ0.100.14Ϯ0.0377.3Xc
0.63Ϯ0.170.34Ϯ0.1051.0S.sanguinis ATCC 105560.26Ϯ0.060.12Ϯ0.0253.9S.oralis
ATCC 105570.67Ϯ0.120.25Ϯ0.0362.7S.gordonii ATCC 10558
0.73Ϯ0.04
0.24Ϯ0.08
66.5
a
Expressed as the reduction of A 550after 2h.Values are the means ϮS.D.of triplicate assays.b
Percent inhibition was calculated as 100ϫ[(a Ϫb /a ],where a is the mean value without lactoferrin (control),and b is the mean value with
lactoferrin.
F I
G .4.Heat stability of bovine lactoferrin (A )and the effect of p
H on the binding of lactoferrin to salivary agglutinin (B ).A ,after bovine lactoferrin (50␮g/ml)was treated at 40to 100°C for 15min,the samples were subjected to surface plasmon resonance analysis.B ,reactions were carried out with salivary lactoferrin (50␮g/ml)in 10m M potassium phosphate buffer (pH 2–8)containing 0.15M NaCl.The binding of lactoferrin to salivary agglutinin is expressed as RU deter-mined by surface plasmon resonance.Values are given as the means ϮS.D.of triplicate assays.
Binding of Bovine Lactoferrin to Salivary Agglutinin
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ferrin inhibited the saliva-induced aggregation of S.mutans cells.The binding of bovine lactoferrin to rPAc,salivary agglu-tinin,and components of salivary agglutinin was examined using surface plasmon resonance.Bovine lactoferrin bound more strongly to salivary agglutinin,especially to high molec-ular mass glycoprotein,than to rPAc,suggesting that bovine lactoferrin may inhibit the interaction between PAc and sali-vary 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 lactof-errin,indicating that the inhibitory effect of lactoferrin is not specific for S.mutans .
The optimal pH for the binding of bovine lactoferrin to sali-vary agglutinin was 4.0,and the stability of lactoferrin to bind to salivary agglutinin was not affected by previous heat treat-ment.The isoelectric point of bovine lactoferrin is ϳ8.0(33).It can be sterilized at high temperatures at pH 4.0without 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-bind-ing capacity is associated with many biological functions (35,36).The lactoferrin preparation used in this study was 19.3%iron-saturated.To examine the role of iron binding in inhibi-tion of S.mutans aggregation,we also prepared apolactoferrin and iron-saturated lactoferrin and assayed them for their abil-ity 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 ,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 experi-ment,Soukka et al.(38)showed that apolactoferrin effectively agglutinates S.mutans cells but not the other bacteria.How-ever,our preliminary studies have shown that all of the three types of lactoferrin did not induce the aggregation of S.mutans cells.2The cause of this discrepancy may be ascribed to differ-ences in strain of S.mutans used or the experimental condition.To identify the saliva-binding region of the lactoferrin mole-cule,we prepared a series of truncated lactoferrin fragments and assayed their effects on the saliva-induced aggregation of S.mutans cells.Our results suggest that lactoferrin residues 473–538play an important role in the inhibition of saliva-induced aggregation of S.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 con-sist 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 cleav-age of lactoferrin and isolated by high performance liquid chro-matography.Here,we prepared truncated lactoferrin frag-ments 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 aggluti-nin remains unclear.The predicted pI value and secondary structure of each lactoferrin fragment were obtained using the DNA software package,DNASIS (Hitachi Software Engineer-ing,Tokyo,Japan).Secondary structure was predicted accord-ing to the method of Chou and Fasman (43).Although all the active fragments containing residues 473–538had acidic pI values,the inactive fragment Lf44also had an acidic pI value (pI ϭ5.2).Therefore,electrostatic interactions do not seem to be involved in agglutinin binding.Furthermore,the inhibitory fragments of lactoferrin did not retain characteristic secondary ctoferricin 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 attrib-uted to the disruption of target cell membranes by the basic residues arrayed along the outside of the lactoferricin B mole-cule (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 mecha-nism 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 ad-sorbed 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
2
M.Mitoma,T.Oho,Y.Shimazaki,and T.Koga,unpublished
data.
F I
G .5.Inhibition of saliva-induced aggregation of S.mutans cells by lactoferrin fragments.S.mutans MT8148cells grown in BHI broth were harvested and resuspended in aggrega-tion buffer.The suspensions were ad-justed to an A 550of ϳ1.5with aggregation buffer.The cell suspensions (1ml)were mixed with 25␮l of whole saliva and 1n M lactoferrin or lactoferrin fragment,and the total volume of the reaction mixture was adjusted to 1.5ml.Aggregation was measured by the reduction in A 550after 2h.Percent inhibition was calculated as 100ϫ[(a Ϫb )/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 ϮS.D.of triplicate assays.*,p Ͻ0.05;**,p Ͻ0.01;***,p Ͻ0.001(compared with DHFR).
Binding of Bovine Lactoferrin to Salivary Agglutinin
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