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Title Bovine lactoferrin region responsible for binding tobifidobacterial cell surface proteins
Author(s) Rahman, Morshedur; Kim, Woan-Sub; Kumura, Haruto;Shimazaki, Kei-ichi
Citation Biotechnology Letters, 31(6): 863-868
Issue Date 2009-06
Right The original publication is available
Type article (author version)
Information 31-6_p863-868.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
The bovine lactoferrin region responsible for binding to
bifidobacteria cell surface protein
Md. Morshedur Rahman, Woan-Sub Kim, Haruto Kumura, Kei-ichi Shimazaki
Dairy Food Science Laboratory, Research Faculty of Agriculture, Hokkaido University,
W-9, N-9, Sapporo, 060-8589, Japan
Corresponding author: Tel: +81-11-706-3642; Fax: +81-11-706-4135
Bovine lactoferrin (bLf) is a multifunctional iron-binding glycoprotein secreted mainly
in milk and other secretory fluids. Bovine lactoferrin is reported to promote the
growth of bifidobacteria and binding of bLf to bifidobacteria cell is thought to be
involved. After separation of bLf half molecule and extraction of surface proteins
from bifidobacteria, binding profiles were observed by immunoblotting. No binding
was appeared when bLf C-lobe was being reacted with cell surface proteins on PVDF
membrane. Conversely, a 50-kDa band was appeared when reacted with either intact
bLf or nicked bLf. This result strongly suggests that binding region could be N-lobe.
Moreover, blot, probed with nicked bLf, reacted with anti-lactoferricin antibody also
produced a 50-kDa band that indicates the binding occurred at lactoferricin region of
bLf molecule. Interestingly, despite absence of binding, bLf C-lobe can stimulate the
growth of bifidobacteria.
Lactoferrin (Lf), a multifunctional iron-binding transferrin family glycoprotein is
secreted mainly in milk and other secretory fluids, e.g. tears, saliva etc. and is also
found in the granules of the neutrophils as reviewed by Shimazaki [1]. This 80 kDa
protein is composed of a single polypeptide chain of about 690 amino acid residues and
is folded into two homologous (~ 40% sequence identity) lobes, representing its N- and
C-terminal halves and connected by a short “hinge” peptide of 10-15 residues. Each
lobe has two domains (N1 and N2, C1 and C2) and can bind a single ferric ion
concomitantly with one bicarbonate ion very tightly [2]. There are striking
conservation between these two lobes in respect of their iron retention ability (C-lobe
bind iron more tightly) [3] and biological functions (some functions of Lf are thought to
be involved in the N-lobe) [4, 5]. N- and C-lobes also possess unique binding regions
for microbial membranes [6]. The participation of N-and C-lobes in binding to cell
surface receptors has also been reported [5, 7, 8]. Bovine lactoferrin (bLf) C-lobe is
also reported to promote the contractile activity of collagen gels more prominently than
native bovine lactoferrin or it’s N-lobe [9].
Bifidobacteria, one of the predominant bacterial groups that exist in the human gut
and play important roles in maintaining human health throughout the life span of the
individual [10], are appeared to require iron for their growth and apparently shown to
produce no siderophores [11]. Studies also have shown that Lf may indeed provide
iron to bifidobacteria [12]. In contrast, Petschow et al. [13] suggested that growth
promotion of Bifidobacterium spp. in vitro is independent of the iron saturation level of
Lf and binding of Lf to bifidobacterial cells may be involved. Lactoferrin binding
proteins have been identified in many Gram-positive and Gram-negative bacteria but
most of their functional roles have not been extensively and definitively determined
[14]. We also previously reported lactoferrin binding protein in bifidobacteria [15, 16].
It would be valuable to identify the bLf region responsible for binding to bifidobacteria
in order to elucidate the molecular analysis of bLf effects on bifidobacteria growth.
Consequently, the main aim of this study was to identify the binding profiles of bLf half
molecule with surface proteins extracted from bifidobacteria.
Materials and Methods
Two strains of bifidobacteria (B. infantis JCM 7007 and B. longum JCM 7054) were
used in this study. Strains were purchased from Japan Collection of Microorganisms
(JCM). All strains were maintained as frozen stocks at – 80ºC in sterile MRS broth
(Merck, Darmstadt, Germany) containing 20% glycerol and 0.05% L-cysteine.HCL.
For further use, each bacterium was reactivated by two consecutive subcultures in MRS
broth containing 0.05% L-cysteine.HCL under anaerobic condition at 37ºC.
Separation of bLf half molecule
Bovine lactoferrin half molecule was separated according to Shimazaki et al. [17].
Lactoferrin was kindly supplied by Morinaga Milk Company Ltd. (Zama, Japan) as
lyophilized form and then iron-saturated as described by Shimazaki and Hosokawa [18].
Partial proteolysis of bLf by trypsin occurred in 0.05 M Tris-HCl buffer, pH 7.8,
containing 0.02 M CaCl2 at 37°C for 2 h. The tryptic digestion mixture of bLf was
applied on a Carboxymethyl Toyopearl 650 (Tosoh, Tokyo) column in the refrigerator.
The unabsorbed parts were washed out with 0.08 M sodium phosphate buffer (pH 6.8)
and the absorbed parts were then eluted with 0.08 M sodium phosphate buffer (pH 7.4)
containing 0.5 M NaCl. Samples from different peaks were collected and dialyzed to
remove salt. After freeze-drying, samples were stored at 4C for further analysis.
Collected samples were also analyzed by sodiumdodecylsulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE) and the bLf C-lobe was recognized by Western blotting
using mouse anti-lactoferrin C-lobe antibody.
Extraction of bifidobacteria cell surface proteins
Bifidobacteria cell surface proteins were extracted as described by Fang and Oliver
(19) and Almeida et al. (20). Bifidobacteria strains were grown in MRS containing
0.05 % L-cysteine.HCl under anaerobic condition for 16 h at 37°C. Bacterial cells
were harvested and washed three times with sterile phosphate buffered saline (PBS, pH
7.4) by centrifugation at 4,000 × g for 10 min. Cell surface proteins were extracted by
incubating with 0.2 % SDS (30 mg moist weight of cell pellets per 100 L of 0.2 %
SDS (w/v) for 1 h at 37°C with intermittent mixing). Extraction mixtures were
centrifuged at 12,000 × g for 10 min and the supernatants (cell surface proteins) were
collected and stored at -20°C for further analysis.
Identification of binding region of bLf to bifidobacteria cell surface protein
Binding region of bLf to bifidobacteria surface proteins was identified by
immunoblotting. The extracted surface proteins from bifidobacteria were separated by
SDS-PAGE (10%) according to Laemmli [21] and were either stained with coomassie
brilliant blue (CBB) R-250 or transferred onto polyvinylidene-difluoride (PVDF)
membrane. The blots were then blocked for 90 min with 3% bovine serum albumin
(BSA) dissolved in PBS containing 0.5% (v/v) Tween 20 (PBST). After removal of
excess amount of blocking reagent, the blots were either probed with intact bLf, bLf
C-lobe or nicked bLf (5 g/mL) for 6 h at 4ºC. After five 15-min washes with PBST,
blots were further probed for overnight at 4ºC with either rabbit anti-bLf antibody
(Fujirebio, Inc., Tokyo) or mouse anti-lactoferricin antibody [22] (in the case of nicked
bLf only) at a dilution of 1:5000 . After five 15-min washes with PBST, blots were
then incubated for 1 h at room temperature with horseradish peroxidase (HRP)
conjugated either goat anti-rabbit or anti-mouse IgG (Wako chemicals, Tokyo) diluted
1/5000. After a final five 15-min washes with PBST, the activity of HRP on blots was
visualized using 3,3`-diaminobenzidinetetrahydrochloride (DAB) as substrate.
Effects of bLf C-lobe on the growth of Bifidobacterium strains
Bifidobacterium strains were grown under anaerobic condition in MRS broth
(Merck, Darmstadt, Germany) containing 0.05% L-cysteiene.HCl at 37C with the
addition of bLf C-lobe or without adding protein (control). Protein solution was
prepared by dissolving in sterilized PBS (pH 7.2) followed by filter sterilization (pore
size 0.20 m). The protein concentration was estimated by spectrophotometric
analysis (A280:A465) using extinction coefficient A2801%,1cm = 15.1 for holo-type and 12.7
for apo-type bLf as reviewed by Shimazaki (1). Two-fold serially diluted protein
solution was added into fresh medium to achieve a final concentration of 4, 2, 1, 0.5 or
0.25 mg/ml. The medium was then inoculated with reactivated Bifidobacterium strain.
For control cultures, PBS was added instead of protein solution. After 16 h incubation
under anaerobic conditons, bacterial growth was monitored spectrophotometrically at
660 nm with 10 times dilution of the cultured medium. The effect was expressed as
relative growth promotion level (%) and calculated using the formula as described by
Saito et al. [23]:
Results are given as mean relative growth promotion level (%) of triplicate assays.
Differences among the means were determined by Duncan’s Multiple Range Test
(DMRT) and P < 0.05 was considered statistically significant.
Results and Discussion
Bovine lactoferrin half molecule was separated by generating bLf fragments (lane 1,
Fig. 1b) with trypsin, which was then applied on Carboxymethyl Toyopearl 650 (Tosoh,
Tokyo) column. As shown in Fig. 1a, the unabsorbed parts (peak-1) had an estimated
molecular mass of around 43 kDa (lane 3, Fig. 1b) and were recognized as bLf C-lobe
by western blot (Fig. 1c). Remaining fragments were eluted with peak-2 and -3 as
shown in Fig. 1a. SDS-PAGE analysis showed multiple bands for peak-2 (lane 4, Fig.
1b) whereas two distinct band with an estimated molecular mass of around 52 and 36
kDa, respectively were observed for peak-3. The fractions eluted as peak-3 was
termed as ‘nicked bLf’ in this study. Theoretically this 36 kDa band represents 80% of
N-lobe and 52 kDa represents fragment containing entire C-lobe and a part of N-lobe.
A summary of bLf half molecule separation is shown in Fig. 1d.
Although, we reported previously bLf binding protein in the membrane associated
fraction of bifidobacteria [15, 16]; recently, we detected and purified bLf-binding
protein (bLf-BP) in the surface proteins of bifidobacteria (data yet to be published) and
the estimated molecular weight is different from that of our previous result [15, 16].
This may be caused by the difference of extraction method from bacteria. The region
of bLf responsible for binding to bifidobacteria cell surface proteins was evaluated by
immunoblotting as shown in Fig. 2. Bifidobacteria cell surface proteins were extracted
and analyzed by SDS-PAGE (Fig 2 A). After transfer proteins onto PVDF membrane
and probed with bovine lactoferrin C-lobe resulting no bands (Fig. 2 C) whereas around
a 50 kDa band was appeared when probed either with intact bovine lactoferrin or nicked
bovine lactoferrin (Fig. 2 B and D). Analogous band of 50-kDa was also appeared
when blot, probed with nicked bLf, was reacted with mouse anti-lactoferricin
(monoclonal) antibody.
The effect of bLf C-lobe on the growth of bifidobacteria in MRS medium was
investigated, and the results are shown in Fig. 3. The concentration of bLf C-lobe was
adjusted to 0.25, 0.5, 1.0, 2.0 or 4.0 mg/mL and the relative growth promotion level was
expressed as the ratio of the absorbance in the presence of bLf C-lobe to the control
absorbance value after 16 h of cultivation. A statistically significant (P < 0.05)
dose-dependent growth stimulating effect by bLf C-lobe was observed for both the
strain tested. However, no statistical differences was found between 0.25 and 0.5
mg/ml and 1.0 and 2.0 mg/ml concentration in B. longum and between 0.5 and 1.0
mg/ml concentration in B. infantis. It should be noted that no inhibitory effect was
observed even at high concentration (4 mg/ml). Comparison of growth responses
between two strains showed a significant difference (P < 0.05) at each concentration as
indicated by asterisks mark in Fig. 3.
The present findings indicate that not bLf C-lobe but may be N-lobe bind with
bifidobacteria cell surface proteins. In Moraxella and Neisseria spp.,
lactoferrin-binding proteins are reported to play role in iron acquisition from lactoferrin
[24] and binds to both domains of the human lactoferrin C-lobe [25]. This different
result may be due to the differences in characteristics between Moraxella and Neisseria
spp. and of Bifidobacterium spp. Studies have shown that bifidobacteria needs iron for
their growth, but iron uptake mechanism of bifidobacteria is not clear yet. Through
binding, utilization of iron from Lf by bifidobacteria may be one possible mechanism
behind the Lf growth stimulation effects on bifidobacteria. Since Lf N-lobe site is
reported to bind iron more weakly than C-lobe [3], it can be explained that
bifidobacteria binds with this site of Lf as a means of iron acquisition. However, this
explanation seems not to be reasonable because growth of bifidobacteria was also
stimulated by bLf C-lobe in a dose-dependent fashion (Fig. 3), even bLf C-lobe did not
bind with bifidobacteria (Fig. 2 C). The present findings suggest several possible
explanations. The C-lobe used in the study was being iron-saturated; as a result one
possible explanation is that growth of bifidobacteria was promoted by utilizing this iron,
and the iron uptake mechanism of bifidobacteria is not related with the binding with
iron-binding protein. This idea is supported by Miller-Catchpole et al. [12] who
studied effect of C-terminal fragment of human latoferrin on the growth of
Bifidobacterium breve. Another explanation is bifidobacteria may hydrolyze protein
by secreting enzymes and the resulting peptides may promote the growth. Liepke et al.
[26] identified bifidogenic peptides from human milk in which lactoferrin-derived
peptides were being reported.
The binding ability of nicked bLf strongly suggests the binding site as bLf N-lobe
(Fig. 3 D). In addition, since bLf-BP was appeared when we used anti-lactferricin
antibody (Fig. 3 E), it can be said that binding occurred at lactoferricin region.
Binding with N-lobe of lactoferrin especially at lactoferricin region generates another
possible explanation. Lactoferrin is known to have bactericidal, fungicidal, and
antiviral activity as well as antitumor, anti-inflammatory and immunoregulatory
properties as described in several review articles [27, 28, 29, 30]. Most of these
activities reside in the N-domain of lactoferrin [31]. This domain is also termed as
antimicrobial domain due to release of lactoferricin, a more potent antimicrobial peptide,
by pepsin digestion [6, 32] and lactoferrampin, a second stretch of N1 domains reported
as another novel antmicrobial peptide [33]. Therefore, bifidobacteria may make
protection against antimicrobial activity of lactoferrin by binding with this domain.
This explanation is supported by the lactoferrin behavior towards bifidobacteria growth
that shows lactoferrin does not inhibit the growth of bifidobacteria rather stimulates.
Lactoferrin is composed of two homologous lobes of which N-lobe is termed as
antimicrobial region and C-lobe is reported to bind iron more tightly. The bovine
lactoferrin region responsible for binding with bifidobacteria surface protein was
suggested to be N-lobe. Although no binding was appeared with bovine lactoferrin
C-lobe, bifidobacteria showed growth responses against C-lobe in a dose-dependent
fashion. This result indicates that bifidobacteria either utilize iron from C-lobe by
other mechanism rather than binding with protein or may hydrolyze protein by secreting
enzymes and the resulting peptides may promote the growth. Binding with nicked
bovine lactoferrin suggests that, since bovine lactoferrin N-lobe has been recognized as
antimicrobial domain, bifidobacteria may make protection against antimicrobial activity
of lactoferrin by binding with this domain.
The authors express their most sincere appreciation to Miss Yokoyama for separating
bovine lactoferrin half molecule that greatly facilitated this study.
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Figure legends
Fig. 1 Separation profiles of bovine lactoferrin (bLf) half molecule. Tryptic digestion
mixture of bLf was applied on a Carboxymethyl Toyopearl 650 column (a). The
unabsorbed parts (peak-1) were washed out with 0.08 M sodium phosphate buffer (pH
6.8) and the absorbed parts were eluted (peak-2 and -3) with 0.08 M sodium phosphate
buffer (pH 7.4) containing 0.5 M NaCl. Tryptic digestion mixture of bLf (lane-1),
intact blf (lane-2), elutes from peak-1 (lane-3), peak-2 (lane-4) and peak-3 (lane-5) were
analyzed by SDS-PAGE (b). The unabsorbed parts (peak-1) were recognized as bLf
C-lobe by western blot (c). Summary of separation profile is shown in a tabular form
Fig. 2 Identification of bovine lactoferrin (bLf) region responsible for binding with
surface proteins of bifidobacteria. Cell surface proteins, extracted from B. longum
JCM 7054 (lane 1) and B. infantis JCM 7007 (lane 2) were analysed by SDS-PAGE (A).
Bovine lactoferrin-binding protein (bLf-BP) was recognized by western blot probing
either with intact bLf (B), bLf C-lobe (C) or nicked bLf (D) followed by further probing
with rabbit anti-bLf antibody. Probing with mouse anti-lactoferricin antibody in
nicked bLf (E) was also carried out. The western blot has the same arrangement of
lanes as in the SDS-PAGE. Absence of any band while probing with bLf C-lobe (C)
and presence of around a 50 kDa band while probing with nicked bLf (D) indicate the
responsible binding region could be N-lobe. Probing with monoclonal antibody also
indicates that the lactoferricin region could be the binding sites. M indicates
prestained protein markers that were used to estimate the molecular weights.
Fig. 3 In vitro effects of bovine lactoferrin (bLf) C-lobe on the growth of
Bifidobacterium strains. Bacteria were grown in MRS medium with or without
addition of bLf C-lobe at various concentrations. Relative growth promotion level was
expressed as the ratio of the absorbance value at 660nm in the presence of bLf C-lobe to
the control absorbance value after 16 h of cultivation at 37C under anaerobic condition.
The average absorbance value of control was 0.36 for B. longum and 0.5 for B. infantis.
The experiment was conducted three times in triplicate. Results are the average of these
triplicate assays. Same letters on the bars indicate no statistical differences whereas
different letters indicate significant differences (P < 0.05) between effects of different
concentrations on the growth of respective strain. Asterisks indicate the statistical
differences (P < 0.05) between growth responses of bacterial strains at specific
1 2 3 4 5 M
Peak-1 3 1 45 bLf C-lobe
Peak-2 4 multiple Not estimated No status
Peak-3 5 2 52
Nicked bLf
Status of
Peak number. in
Lane number
Number of
mass (kDa)
(b) (c)
0 50 100 150 200
Time (min)
bs or ba nc e at 2
n m 0
on du ct iv ity
m S
/c m peak-1
(A) (B) (D)(C)
1 2 1 2 1 2 1 2 1 2M
0.25 mg/ml 0.5mg/ml 1.0 mg/ml 2 mg/ml 4 mg/ml
el at iv e gr ow th p ro m ot io n le ve l (
B. longum JCM 7054 B. infantis JCM 7007
a a ab b c a ab b c d *

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