The bovine lactoferrin region responsible for binding to bifidobacteria cell surface protein

Content Provider	Semantic Scholar
Author	Rahman, Morshedur Kim, Woan-Sub Kumura, Haruto Shimazaki, Kei-Ichi
Copyright Year	2017
Abstract	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. Introduction 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 Nand 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]. Nand 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 Bacteria 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 A280 = 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 on
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