C-lobe Of Bovine Lactoferrin Research Articles

Heterologous expression of bovine lactoferrin C-lobe in Bacillus subtilis and comparison of its antibacterial activity with N-lobe

The natural concentration of bovine lactoferrin C-lobe is low and its separation by proteolytic enzyme digestion is difficult. Here, we expressed the codon-optimized fragment of C-lobe on plasmid pMA0911 with the Pveg promoter in Bacillus subtilis 168 at 20 °C. The yield was 7.5 mg/L, and 90.6% purity was achieved using ammonium sulfate precipitation, Ni–NTA and molecular exclusion. The C-lobe at 10 mg/mL completely inhibited cell growth of Escherichia coli JM109 (DE3) and Pseudomonas aeruginosa CGMCC 1.6740, and 48.4% of growth of Staphylococcus aureus CGMCC 1.282, the result is similar to that of 200 ng/mL N-lobe. The minimum inhibitory concentrations of C-lobe were 4, 8 and 16 mg/mL, while those of N-lobe were 128, 256 and 512 μg/mL for E. coli, P. aeruginosa and S. aureus, respectively. This is the first report on bovine lactoferrin C-lobe expression and the comparative resistance of the recombinant N- and C-lobes in a food-safe strain of B. subtilis. Our findings offer the potential to study the structure–function relationship of the N- and C-lobes recombinantly produced in the same host.

Investigation on side-product formation during the synthesis of a lactoferrin-derived lactam-bridged cyclic peptide.

Bovine lactoferrin C-lobe is able to prevent both influenza virus hemagglutination and cell infection. In particular, it was demonstrated that the fragment 418SKHSSLDCVLRP429 is a potent antiviral peptide. Therefore, we tried to increase the stability of this fragment through side-chain lactam cyclization of the peptide, S[KHSSLD]CVLRP (1). However, classic strategy involving solid-supported cyclization of the linear precursor, containing orthogonal allyl/alloc-based protection for the key amino and carboxyl residues, did not provide the desired cyclic peptide. Here, we report the identification of problematic stretches during the sequence assembly process and the optimization of the different parameters involved in the construction of 1. Results indicated a significant influence of β-protecting group of both aspartic acid and adjacent cysteine residues on the formation of side products. Therefore, the identification of suitable β-protecting groups of these residues allowed us to optimize the synthesis of designed lactam-bridged cyclic peptide.

Lactoferrin-derived Peptides Active towards Influenza: Identification of Three Potent Tetrapeptide Inhibitors

Bovine lactoferrin is a biglobular multifunctional iron binding glycoprotein that plays an important role in innate immunity against infections. We have previously demonstrated that selected peptides from bovine lactoferrin C-lobe are able to prevent both Influenza virus hemagglutination and cell infection. To deeper investigate the ability of lactoferrin derived peptides to inhibit Influenza virus infection, in this study we identified new bovine lactoferrin C-lobe derived sequences and corresponding synthetic peptides were synthesized and assayed to check their ability to prevent viral hemagglutination and infection. We identified three tetrapeptides endowed with broad anti-Influenza activity and able to inhibit viral infection in a concentration range femto- to picomolar. Our data indicate that these peptides may constitute a non-toxic tool for potential applications as anti-Influenza therapeutics.

Inhibitory Effects of Synthetic Peptides Containing Bovine Lactoferrin C-lobe Sequence on Bacterial Growth.

Lactoferrin is a glycoprotein with various biological effects, with antibacterial activity being one of the first effects reported. This glycoprotein suppresses bacterial growth through bacteriostatic or bactericidal action. It also stimulates the growth of certain kinds of bacteria such as lactic acid bacteria and bifidobacteria. In this study, Asn-Leu-Asn-Arg was selected and chemically synthesized based on the partial sequences of bovine lactoferrin tryptic fragments. Synthetic Asn-Leu-Asn-Arg suppressed the growth of Pseudomonas fluorescens, P. syringae and Escherichia coli. P. fluorescens is a major psychrotrophic bacteria found in raw and pasteurized milk, which decreases milk quality. P. syringae is a harmful infectious bacterium that damages plants. However, synthetic Asn-Leu-Asn-Arg did not inhibit the growth of Lactobacillus acidophilus. It is expected that this synthetic peptide would be the first peptide sequence from the bovine lactoferrin C-lobe that shows antibacterial activity.

Structure of iron saturated C-lobe of bovine lactoferrin at pH 6.8 indicates a weakening of iron coordination.

The bilobal lactoferrin is an approximately 76 kDa glycoprotein. It sequesters two Fe(3+) ions together with two CO(3)(2-) ions. The C-terminal half (residues, Tyr342-Arg689, C-lobe) of bovine lactoferrin (BLF) (residues Ala1-Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact BLF were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 Å resolution and refined to R(cryst) and R(free) factors of 0.205 and 0.273, respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 Å for their C(α) atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 Å(2). The distance of 2.9 Å observed for the coordination bond between Fe(3+) ion and N(e2) of His595 appeared to be considerably longer than the normally observed values of 1.9-2.2 Å. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1-2.3 Å. Based on the present structure of iron saturated C-lobe, it may be stated that His595 is the first residue to dissociate from ferric ion when the pH is lowered.

Structure of the iron-free true C-terminal half of bovine lactoferrin produced by tryptic digestion and its functional significance in the gut.

Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N- and C-terminal molecular halves with the ability to bind two Fe(3+) ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apo-lactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation.

Bovine Lactoferrin Structures Promoting Corneal Epithelial Wound Healing In Vitro

To use an in vitro alkali-induced wound model to identify structures of bovine lactoferrin (BLF) that contribute to the promotion of human corneal epithelial healing. BLF N-lobe and C-lobe were separated using limited proteolysis and purified by preparative chromatography. Isoforms of BLF were separated according to serine protease activity. Catalytic activities of isoforms and lobes were quantified by hydrolysis of a synthetic serine protease substrate. The promotion of healing by cognate moieties, lactoferricin-B, BLF isoforms, and BLF in various forms-iron-free, iron-saturated, deglycosylated, zwitterionic detergent exposed, chaotrope denatured, disulfide reduced-was assessed on alkali wounding of confluent monolayers of human corneal epithelial cells. The C-lobe of BLF (6.4-128 μM) promoted greater wound healing than native-BLF or N-lobe. BLF (12.8 μM) promoted wound closure in an iron-free, iron-bound, or deglycosylated state or after exposure to zwitterionic detergent. Healing was not stimulated by chaotropically denatured or disulfide reduced BLF (12.8 μM) or by lactoferricin-B (12.8 μM). Proteolytically active BLF (0.6 μM) promoted wound closure at a lower concentration than proteolytically inactive BLF (12 μM). This proteolytic activity was localized to the N-lobe. The C-lobe is the primary promoter of BLF-stimulated corneal epithelial wound closure in vitro and is effective at concentrations ≥6.4 μM. Increased healing from BLF occurs with the native conformation and is unaffected by glycosylation or iron saturation. To a lesser extent, proteolytic activity of the N-lobe also improves healing rates. The BLF C-lobe may be a novel treatment for corneal lesions with delayed healing.

Expression of Bovine Lactoferrin C-lobe inRhodococcus erythropolisand Its Purification and Characterization

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 degrees C with shaking, and expression of the rBLF C-lobe was induced by adding 1 microg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.

Crystal Structure of a Proteolytically Generated Functional Monoferric C-lobe of Bovine Lactoferrin at 1.9 Å Resolution

This is the first crystal structure of a proteolytically generated functional C-lobe of lactoferrin. The purified samples of iron-saturated C-lobe were crystallized in 0.1 M Mes buffer (pH 6.5) containing 25% (v/v) polyethyleneglycol monomethyl ether 550 M and 0.1 M zinc sulphate heptahydrate. The X-ray intensity data were collected with 300 mm imaging plate scanner mounted on a rotating anode generator. The structure was determined by the molecular replacement method using the coordinates of the C-terminal half of bovine lactoferrin as a search model and refined to an R-factor of 0.193 for all data to 1.9 Å resolution. The final model comprises 2593 protein atoms (residues 342–676 and 681–685), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), one Fe 3+, one CO 3 2−, two Zn 2+ and 230 water molecules. The overall folding of the C-lobe is essentially the same as that of C-terminal half of bovine lactoferrin but differs slightly in conformations of some of the loops and reveals a number of new interactions. There are 20 Cys residues in the C-lobe forming ten disulphide links. Out of these, one involving Cys481-Cys675 provides an inter-domain link at 2.01 Å while another Cys405-Cys684 is formed between the main C-lobe 342-676 and the hydrolyzed pentapeptide 681-685 fragment. Six inter-domain hydrogen bonds have been observed in the structure whereas only four were reported in the structure of intact lactoferrin, although domain orientations have been found similar in the two structures. The good quality of electron density has also revealed all the ten oligosaccharide units in the structure. The observation of two metal ions at sites other than the iron-binding cleft is another novel feature of the present structure. These zinc ions stabilize the crystal packing. This structure is also notable for extensive inter-molecular hydrogen bonding in the crystals. Therefore, the present structure appears to be one of the best packed crystal structures among the proteins of the transferrin superfamily.

Fine Structures of Epitopic Sites in Human and Bovine Lactoferrin Recognized by Anti-bovine Lactoferrin C-Lobe Monoclonal Antibody

The reactivity towards the monoclonal antibody (MAb) against bovine lactoferrin C-lobe was compared with six members of the transferrin family of proteins by ELISA and dot-blotting methods. This MAb recognizes WNIPMGL (467-473) of the bovine lactoferrin sequence. Human lactoferrin, Korean native goat lactoferrin and bovine transferrin showed weak reactivity with anti-C-lobe MAb. Human transferrin and ovotransferrin did not react with this antibody. As human lactoferrin in the denatured state showed reactivity with the antibody by ELISA, the configurational features of bovine lactoferrin and human lactoferrin were compared. It is observed that human lactoferrin epitopic site is covered with the bulky group and becomes exposed after denaturation.

Structural and immunochemical studies on bovine lactoferrin fragments.

Lactoferrin (Lt) is a metal-binding protein found in milk and other secretory fluids and also in blood. It shows multifunctional properties but the mechanism of developing its function in living systems has not been resolved yet. It is known to exert bacteriostatic effects due to its ability to bind environmental iron. Moreover, apo-lactoferrin has been shown to bind to microbial membranes and causes the direct destruction of microorganisms. Other biological functions attributed to lactoferrin include roles in modulation of the inflammatory response, activation of the immune system, and control of myelopoiesis or cell growth. This molecule is constructed with N- and C-lobes, each of which is composed of 3 domains1. The function of each lobe has been studying and there are certain differences. The biologically significant function has been found mainly in N-lobe. For the aids of resolving their functional analysis, the authors prepared the monoclonal antibodies (mAb) against N-lobe and C-lobe of bovine lactoferrin. To prepare the mAb specific to N-lobe, we used lactoferricin® B (bLfcin, an anti-microbial peptide isolated from N-lobe2) as an antigen3. In this paper, the characterization of the mAb against lactoferrin fragments has been examined and the structure of the mAb-recognition site on lactoferrin molecule was identified.

Monoclonal antibody against bovine Lactoferricin and its epitopic site.

Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".