From the Department of Preventive Dentistry, Kyushu University Faculty of Dental Science, Fukuoka 812-8582, Japan
Received for publication, February 15, 2001, and in revised form, March 6, 2001
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ABSTRACT |
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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 473-538
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.
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
(PAc)1 of 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 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). 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.
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). Streptococcus
sanguinis ATCC 10556, Streptococcus oralis
ATCC 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
(20) for Escherichia 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
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 (9).
Recombinant PAc (rPAc)--
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 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 (Shimadzu, Kyoto, Japan).
Aggregation Assay--
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).
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
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 (27).
Heat Treatment--
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.
Bovine Lactoferrin Fragments--
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 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 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 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-
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 1 M 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.
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 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, 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 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.
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 a single band
(data not shown). The N-terminally truncated 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 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 (3, 9). Gong
et al. (32) also showed that salivary film on hydroxyapatite contains a complex of macromolecular protein enriched in sIgA and
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 (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 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. (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 experiment, Soukka et al.
(38) 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. mutans
cells.2 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 suggest that lactoferrin residues 473-538 play an
important role in the inhibition of saliva-induced aggregation of
S. mutans. Other fragments lacking these residues, such as
LfN (residues 1-344), 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) (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 473-538
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 (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 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 (39). 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 473-538 of bovine lactoferrin play an important role in the interaction of lactoferrin with salivary agglutinin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-casein,
-casein,
-casein,
lactalbumin, lactoferrin, and lactoperoxidase were purchased from
Sigma. Bovine
-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 (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 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. (24), with bovine serum albumin as a standard.
-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 eluate was less than 0.01. The
fusion proteins were eluted with buffer B containing 250 mM imidazole.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 ( ) and for their inhibitory effect on the
aggregation of S. mutans cells (
).
-
-
,
NaCl gradient.
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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
-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).
-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. 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).
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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 ~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.
Effects of various milk components on the saliva-induced aggregation of
S. mutans MT8148 cells
Effect of lactoferrin on the saliva-induced aggregation of
streptococcal cells
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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 2-8)
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.
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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 ~1.5 with aggregation buffer. The cell
suspensions (1 ml) were mixed with 25 µl of whole saliva and 1 nM 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).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
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ACKNOWLEDGEMENTS |
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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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FOOTNOTES |
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* This work was supported in part by Grants-in-aid for Developmental 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 publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely 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.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101459200
2 M. Mitoma, T. Oho, Y. Shimazaki, and T. Koga, unpublished data.
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ABBREVIATIONS |
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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.
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