Effects of lactoferrin on rat dermal interstitial fluid pressure (Pif) and in vitro endothelial barrier function

We recently demonstrated that intravenous (i.v.) injection of the iron‐binding protein lactoferrin (Lf) followed by antilactoferrin (aLf) antibodies or iron‐saturated Lf alone increased albumin extravasation in vivo in several tissues including skin. Increased driving pressure for blood‐tissue exchange or direct effects of Lf on the endothelial barrier are possible mechanisms. We therefore, firstly, measured interstitial fluid pressure (P_if) in dermis of rats given 1 mg Lf i.v. followed 30 min later by aLf or saline and circulatory arrest 1 or 5 min thereafter and compared with controls. Secondly, transmonolayer passage of Evans blue labelled albumin (EB‐albumin) was evaluated in porcine pulmonary artery endothelial cells exposed to iron‐free or iron‐saturated Lf (both 100 μg mL^–1) in the absence and presence of 0.5 mM hydrogen peroxide. P_if increased significantly at 11–30 min following Lf to +2.1 ± 0.3 and +1.7 ± 0.2 mmHg at 11–20 and 21–30 min, respectively, compared with +0.1 ± 0.2 mmHg before Lf (P < 0.05, n=25). Endothelial transmonolayer passage of EB‐albumin during 3 h was not affected by iron‐free or iron‐saturated Lf neither in the absence nor presence of hydrogen peroxide that increased passage 3.5 times compared with controls. In conclusion, Lf‐induced increase in albumin extravasation in rat skin is not explained by changes in P_if (because Lf raised P_if significantly) or direct effects of Lf on the endothelial barrier.     

Lactoferrin and anti-lactoferrin antibodies: effects of ironloading of lactoferrin on albumin extravasation in different tissues in rats

Lactoferrin is a cationic iron-binding protein, which is released from activated neutrophils in concert with reactive oxygen species. In vitro, lactoferrin has both anti- and proinflammatory effects; many of them dependent on iron-binding. In vivo, only iron-free lactoferrin reduced inflammatory hyperpermeability in the lung. We therefore examined whether 1 mg iron-free (Apo-Lf) or iron-saturated lactoferrin (Holo-Lf) alone or followed by anti-lactoferrin antibodies (aLf) affected permeability evaluated by extravasation of radiolabelled bovine serum albumin (CBSA) in different tissues of anaesthetized rats. Fifteen minutes after i.v. injection of Lf, aLf or saline was given and circulatory arrest was induced 20 min thereafter. Measurements were performed in control, after Apo-Lf, Holo-Lf, Apo-Lf + aLf, Holo-Lf + aLf and aLf alone (n=6-8 in each group). No intergroup differences were found for plasma volume and haematocrit at the start and end of the 37 min extravasation period or for total tissue water in any of the six different tissues studied, excluding larger transcapillary fluid shifts. However, increases in CBSA were seen without differences in tissue intravascular volume. Iron-free lactoferrin and aLf alone did not change CBSA significantly. Iron-saturated lactoferrin significantly increased CBSA in skin (neck), trachea and left ventricle of the heart to 249 +/- 9, 284 +/- 16 and 160 +/- 7% of control, respectively. When followed by aLf, both Apo- and Holo-Lf increased CBSA significantly in four and five of the tissues studied, respectively. However, no significant effect was seen for Holo-Lf + aLf compared with Holo-Lf alone. In conclusion, iron-saturated, but not iron-free lactoferrin increased CBSA, whereas antilactoferrin increased CBSA compared with lactoferrin alone only when following iron-free lactoferrin.     

Effects of lactoferrin on rat dermal interstitial fluid pressure (Pif) and in vitro endothelial barrier function.

We recently demonstrated that intravenous (i.v.) injection of the iron-binding protein lactoferrin (Lf) followed by antilactoferrin (aLf) antibodies or iron-saturated Lf alone increased albumin extravasation in vivo in several tissues including skin. Increased driving pressure for blood-tissue exchange or direct effects of Lf on the endothelial barrier are possible mechanisms. We therefore, firstly, measured interstitial fluid pressure (Pif) in dermis of rats given 1 mg Lf i.v. followed 30 min later by aLf or saline and circulatory arrest 1 or 5 min thereafter and compared with controls. Secondly, transmonolayer passage of Evans blue labelled albumin (EB-albumin) was evaluated in porcine pulmonary artery endothelial cells exposed to iron-free or iron-saturated Lf (both 100 microg mL-1) in the absence and presence of 0.5 mM hydrogen peroxide. Pif increased significantly at 11-30 min following Lf to +2.1 +/- 0.3 and +1.7 +/- 0.2 mmHg at 11-20 and 21-30 min, respectively, compared with +0.1 +/- 0.2 mmHg before Lf (P < 0.05, n=25). Endothelial transmonolayer passage of EB-albumin during 3 h was not affected by iron-free or iron-saturated Lf neither in the absence nor presence of hydrogen peroxide that increased passage 3.5 times compared with controls. In conclusion, Lf-induced increase in albumin extravasation in rat skin is not explained by changes in Pif (because Lf raised Pif significantly) or direct effects of Lf on the endothelial barrier.     

Antioxidant properties of lactoferrin from human milk

Chemiluminescent methods showed that human lactoferrin more effectively inhibits free radical processes in the Fenton reaction than histidine and mannitol, usual free radical scavengers. Human lactoferrin added to yolk lipoprotein suspension in the presence of rhodamine 6G reduces the intensity of fast flash of Fe²⁺-induced chemiluminescence by 37%. Complete saturation of lactoferrin with iron reduces its antioxidant properties by 15.4%, the intensity of chemiluminescence being below the control by 25.7%. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 5, pp. 523–525, May, 1999     

Recombinant pseudoadenovirus nanostructure with the human lactoferrin gene: Production and study of lactoferrin expression and properties in vivo

The method of homologous recombination in E. coli cells was used to obtain a pseudoadenovirus nanostructure Ad5-lf that ensures the effective expression of the lactoferrin (Lf) gene in a permissive cell system with the high production of recombinant protein, which is similar in some physicochemical and biological properties to native human Lf. It has been shown in vivo that the single intravenous introduction of this construction into mouse or rat organism results in the high expression and long circulation of recombinant human Lf in the blood of experimental animals without any toxic effect. The findings are evidence of the good prospects for using pseudoadenovirus nanostructure Ad5-lf for the prolonged production of recombinant human Lf in human organism.     

Biochemical and immunological properties of lactoferrin binding proteins fromMoraxella (Branhamella) catarrhalis

The Neisseriaceae can acquire iron (Fe) from lactoferrin (Lf) using host-Lf receptors on the bacterial surface. The binding proteins that are proposed to constitute the receptor have been identified by isolation with immobilized Lf. Using CopB-specific monoclonal antibodies and isogenic CopB mutants, we demonstrate that the 84 kDa protein isolated with immobilized human Lf fromMoraxella catarrhalisusing low stringency conditions is CopB, an 84 kDa membrane-spanning protein with similarities to other TonB-dependent outer membrane proteins. Affinity isolation of Lf receptors from a variety ofM. catarrhalisstrains using high stringency conditions revealed a 95 kDa protein migrating slightly faster than LbpA on SDS-PAGE in some strains. Convalescent human antisera from patients infected withM. catarrhalisreacted specifically with this protein, but not LbpA. Proteolysis experiments demonstrated that, unlike LbpA, it was rapidly degraded. The 95 kDa protein, but not LbpA, binds labelled Lf after SDS-PAGE and electroblotting, suggesting the 95 kDa protein is LbpB, the homologue of TbpB. This protein comigrates with LbpA in most strains, which may explain why it had not been previously identified.     

Growth and adsorption of Streptococcus mutans 6715-13 to hydroxyapatite in the presence of lactoferrin

The growth of Streptococcus mutans 6715-13 in a rich medium (Todd Hewitt broth) was drastically reduced by addition of apo-lactoferrin (apo-Lf); this effect was bacteriostatic and reversible by saturation of Lf with iron. The influence of Lf, salivary proteins (SP) and bovine serum albumin (BSA) on the attachment of Streptococcus mutans to hydroxyapatite (HA) was successively investigated. Sorption of Lf, SP, and BSA to HA was dependent on the protein concentration and reached the end-point at about 80 mg of proteins per gram of HA. Similarly, the number of streptococci adsorbed to HA was correlated to the amount of cells available up to at least 10⁷ cells per mg of HA. The adsorption of Lf, SP and BSA on HA reduced the number of attaching S. mutans cells. In particular, SP reduced the adsorption of S. mutans by 30%, whereas pre-coating of HA with apoor iron-saturated Lf resulted in a three orders of magnitude reduction of S. mutans adsorption to HA, as demonstrated by means of different experimental procedures. The powerful adherence-inhibiting effect of apo-Lf together with its noticeable antibacterial activity towards S. mutans points to a biological significance of these phenomena also in vivo.     

Influence of lactoferrin on the entry process of Escherichia coli HB101(pRI203) in HeLa cells

Lactoferrin (Lf) is an iron-binding protein which plays an important role in the host defense systems of different mucosal surfaces including the intestinal mucosa. In the present research the role of apo-Lf and iron-saturated Lf in the invasion process of enteroinvasive bacteria, grown in iron stress or excess, was investigated. As enteroinvasive bacterium, Eschericha coli HB101 strain harboring a plasmid which contains the chromosomal inv gene from Yersinia pseudotuberculosis was utilized. The product of this gene (invasin) enables this microorganism to invade human epithelial cultured cells (HeLa). The results obtained showed that apo-Lf and iron-saturated Lf added at physiological concentration during the infection exerted a significant inhibition of adhesion (3.2 × 10⁵ instead 3.4 × 10⁶ adherent bacteria grown in iron excess; 1.6 × 10³ instead of 2.3 × 10⁴ adherent bacteria grown in iron-limited medium) and internalization (4.0 × 10⁵ instead of 3.7 × 10⁶ internalized bacteria grown in iron excess; 2.1 × 10³ instead 2.8 × 10⁴ internalized bacteria grown in iron-limited medium). It has also been demonstrated that in these experimental conditions Lf binds to HeLa cell membrane as well as to bacterial outer membrane. It is likely that this binding interfere with the early events of interaction between bacteria and eukaryotic cells. This inhibiting effect of Lf on the invasion efficiency of E. coli HB101(pRI203) could be related to the cationic nature of the molecule, although other mechanisms cannot be ruled out.     

Protection of SK-N-MC cells against β-amyloid peptide-induced degeneration using neuron growth factor-loaded liposomes with surface lactoferrin

A liposomal system with surface lactoferrin (Lf) was developed for delivering neuron growth factor (NGF) across the blood–brain barrier (BBB) and improving the viability of neuron-like SK-N-MC cells with deposited β-amyloid peptide (Aβ). The Lf-grafted liposomes carrying NGF (Lf/NGF-liposomes) were applied to a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes (HAs) and to fibrillar Aβ_1-42-insulted SK-N-MC cells. An increase in cholesterol mole percentage enhanced the particle size, absolute value of zeta potential, and physical stability, however, reduced the entrapment efficiency and release rate of NGF. In addition, an increase in Lf concentration increased the particle size, surface nitrogen percentage, NGF permeability across the BBB, and viability of HBMECs, HAs, and SK-N-MC cells, however, decreased the absolute value of zeta potential, surface phosphorus percentage, and loading efficiency of Lf. After treating with Lf/NGF-liposomes, a higher Aβ concentration yielded a lower survival of SK-N-MC cells. The current Lf/NGF-liposomes are efficacious drug carriers to target the BBB and inhibit the Aβ-induced neurotoxicity as potential pharmacotherapy for Alzheimer's disease.     

Plasma lactoferrin levels are decreased in end-stage AIDS patients.

The antimicrobial protein lactoferrin (Lf) is present in plasma and in mucosal secretions. Using ELISA we analysed plasma and saliva of HIV-infected patients, patients with AIDS, and healthy controls for the presence of secreted Lf. The plasma Lf levels of AIDS patients (classification C3) were significantly lower (p < 0.001) as compared to asymptomatic and symptomatic HIV infected patients, or controls. In addition, plasma Lf levels closely correlated with neutrophilic granulocyte counts in the HIV-infected patients. Thus, basal plasma Lf levels are likely the result of Lf release by neutrophilic granulocytes. The Candida titres present in the oral cavity were determined in a part of the HIV-infected patient group. As it appeared, the presence of this opportunistic pathogen always coincided with low levels of salivary Lf levels. We conclude that Lf, as part of the nonspecific immune system, might play an important role in the first line of defense against opportunistic microbial infections in AIDS patients.     

Functional domain of bovine milk lactoferrin which inhibits the adherence of Streptococcus mutans cells to a salivary film

The bovine lactoferrin molecule and relatively long lactoferrin fragments containing residues 473 to 538 strongly inhibited adherence of Streptococcus mutans to saliva-coated hydroxyapatite beads. Each cysteine residue in Lf411 (residues 473 to 538) was replaced by a serine residue, and the mutants Lf411-C481S and Lf411-C532S strongly inhibited S. mutans adherence. These results suggest that the functional domain of lactoferrin that binds to a salivary film lies in residues 473 to 538 and that the region might be concealed by disulfide bond formation between Cys481 and Cys532 in the Lf411 fragment.     

Interaction of lactoferrin with Escherichia coli cells and correlation with antibacterial activity

It has been established that the antimicrobial activity of lactoferrin towards Escherichia coli is enhanced by a direct contact between the protein and the microbial cell and that, in the case of E. coli K-12 strains, an antibacterial activity of lactoferrin unrelated to iron withdrawal is present. Evidence is now reported that lactoferrin binds to surface structures expressed in E. coli K-12 strains grown in either an excess or stress of iron. Under the experimental conditions used, lactoferrin binding both in the apo and in the iron-saturated form yields a maximum of 1.6 × 10⁵ bound molecules/E. coli K-12 cell; the amount of lactoferrin bound does not depend on the expression of the iron-regulated outer membrane proteins. In contrast, lactoferrin does not bind to E. coli clinical isolates. Apo-lactoferrin (at 500 /ml in a chemically defined medium) inhibits the growth of E. coli K-12 strains but not of clinical isolates. These findings suggest that the antibacterial activity of the protein could be associated to its binding to the cell surface.     

Inhibition of poliovirus type 1 infection by iron-, manganese- and zinc-saturated lactoferrin

In this study we investigated the inhibitory activity of different milk proteins on poliovirus infection in Vero cells. Proteins analyzed were mucin, α-lactalbumin, β-lactoglobulin, and bovine and human lactoferrin. Viral cytopathic effect was not prevented by mucin, α-lactalbumin or β-lactoglobulin, whereas the lactoferrins tested were able to inhibit the replication of poliovirus in a dose-dependent manner. Further experiments were carried out in which apo- and native lactoferrin or lactoferrin fully saturated with ferric, manganese or zinc ions were added to the cells during different phases of viral infection. Results obtained demonstrated that all lactoferrins were able to prevent viral replication when present during the entire cycle of poliovirus infection or during the viral adsorption step. Only zinc lactoferrin strongly inhibited viral infection when incubated with the cells after the viral attachment, being the inhibition directly correlated with the degree of zinc saturation. Our results demonstrated that all lactoferrins interfered with an early phase of poliovirus infection; zinc lactoferrin was the sole compound capable of inhibiting a phase of infection subsequent to virus internalization into the host cells. Received: 30 November 1998     

Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated at 37 °C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34±2.2 nmol g^-1 ghosts with a dissociation equilibrium constant (37 °C) K_dm 1.38±0.15 fold K_d of albumin binding. K_dm is temperature independent and ～7–8 nm . Exchange efflux kinetics at 0 °C to buffers of various albumin concentrations ([BSA_y]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450±0.018 and the rate constant of unidirectional flux from inside to outside is 0.067±0.01 s^-1. The rate constant of flux from the extracellular side of the membrane to BSA_y increases with the square root of [BSA_y] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL–BSA complex at 0 °C of 0.0063±0.0003 s^-1. Exchange efflux from ghosts containing four different [BSA_i] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rate coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.     

Serum lactoferrin level as a serologic biomarker for allergic rhinitis

Background Allergic rhinitis (AR) is a very common disease and a risk factor for allergic asthma. The discovery of new biomarkers for the early detection of AR would improve the clinical outcomes and reduce socio‐economic burden. We sought to identify a novel serologic marker for detection of AR using a proteomic approach. Methods To identify the proteins involved in AR, comparative proteomics was applied using nasal lavage fluids (NLFs) taken before and after a nasal provocation test (NPT) with Dermatophagoides pteronyssinus (Dpt) in a subject with AR sensitized to Dpt. The clinical relevance of the identified proteins was evaluated by ELISA using NLFs and sera from the three study groups: Dpt‐sensitive AR; asymptomatic Dpt‐sensitive controls; and non‐atopic healthy controls. The sensitivities and specificities of the candidate proteins for predicting AR were determined using receiver operating characteristic (ROC) curves. Results In proteomic analysis, lactoferrin expression was up‐regulated after NPT. The validation study using ELISA showed a significantly lower serum lactoferrin level in the AR group than those of the other two groups (P<0.05, respectively). To discriminate between subjects with or without AR, the optimal serum cut‐off level of lactoferrin was set at <307 ng/mL using the ROC curve. The sensitivity and specificity for predicting AR were 81.4% and 58%. When combined with serum Dpt‐specific IgE level, the sensitivity and specificity for predicting AR were 76.7% and 79.2%. Conclusion These results suggest that the serum lactoferrin level is associated with the phenotype of Dpt‐sensitive AR, and in combination with the serum Dpt‐specific IgE level, may be a potential serologic marker for early detection of AR. Cite this as: G.‐S. Choi, S.‐Y. Shin, J.‐H. Kim, H.‐Y. Lee, N. S. Palikhe, Y.‐M. Ye, S.‐H. Kim and H.‐S. Park, Clinical & Experimental Allergy, 2010 (40) 403–410.     

Bimodal effects of lactoferrin on proliferation of an interleukin‐2‐dependent cytotoxic t‐lymphocyte cell line

The mechanism for the hypo‐responsiveness of mammary gland mononuclear cells (MGMCs) to mitogen stimulation is poorly understood. It has been hypothesized that hypo‐responsiveness of MGMCs may be due to interference by lactoferrin or other whey proteins present in mammary secretions with lymphocyte proliferation stimulated by interleukin‐2 (IL‐2). To address this hypothesis, an experiment was designed to evaluate proliferation of an IL‐2‐dependent bovine cytotoxic T‐lymphocyte cell line (BT‐2) in the presence of apo‐ and iron‐saturated lactoferrin (APOLF and FELF) and transferrin (APOTF and FETF), immunoglobulin G (IgG) and serum albumin (SA) at concentrations ranging from 0.02 to 2.5 mg ml^‐1. The lowest concentration of APOLF and FELF increased BT‐2 cell proliferation. Conversely, increasing concentrations of LF significantly inhibited BT‐2 cell proliferation. Only the highest concentrations of APOTF, FETF and IgG negatively influenced BT‐2 cell proliferation. Serum albumin had no effect. These data suggest that LF has a dose‐dependent bimodal influence on a bovine IL‐2‐dependent cytotoxic T‐lymphocyte cell line, and high concentrations of LF and other mammary secretion whey proteins may be associated with MGMC hypo‐responsiveness during the non‐lactating period, possibly through interference with IL‐2‐stimulated lymphocyte proliferation.     

Solvent effects on free-radical polymerization, 2. IR and NMR spectroscopic analysis of monomer mixtures of methyl methacrylate and N-vinyl-2-pyrrolidone in bulk and in model solvents

Binary systems of methyl methacrylate (MMA)/N-vinyl-2-pyrrolidone (NVP) and MMA/N-methyl-2-pyrrolidone (NMP) with NMP as saturated model of NVP and of NVP/methyl isobutyrate (MiB) with MiB as saturated model of MMA were investigated by means of IR and NMR spectroscopy. Investigations were carried out at room temperature or at 60°C in CHCl₃ (IR) or CDCl₃ and C₆H₁₂/C₆D₁₂ (NMR). It can be concluded from IR and NMR spectra that the polarity of MMA increases in the presence of NVP and the polarity of NVP decreases in the presence of MMA. Equilibrium constants K of complex formation were determined to: K = 0,169 ± 0,037 L · mol^?1 for MMA/NVP at 30°C, 0,112 ± 0,024 L · mol^?1 for NVP/MiB at 60°C and 0,125 ± 0,030 L · mol^?1 for MMA/NMP at 60°C.     

The role of gray and white matter segmentation in quantitative proton MR spectroscopic imaging

Since the brain's gray matter (GM) and white matter (WM) metabolite concentrations differ, their partial volumes can vary the voxel's ¹H MR spectroscopy (¹H‐MRS) signal, reducing sensitivity to changes. While single‐voxel ¹H‐MRS cannot differentiate between WM and GM signals, partial volume correction is feasible by MR spectroscopic imaging (MRSI) using segmentation of the MRI acquired for VOI placement. To determine the magnitude of this effect on metabolic quantification, we segmented a 1‐mm³ resolution MRI into GM, WM and CSF masks that were co‐registered with the MRSI grid to yield their partial volumes in approximately every 1 cm³ spectroscopic voxel. Each voxel then provided one equation with two unknowns: its i‐ metabolite's GM and WM concentrations C_i^GM, C_i^WM. With the voxels' GM and WM volumes as independent coefficients, the over‐determined system of equations was solved for the global averaged C_i^GM and C_i^WM. Trading off local concentration differences offers three advantages: (i) higher sensitivity due to combined data from many voxels; (ii) improved specificity to WM versus GM changes; and (iii) reduced susceptibility to partial volume effects. These improvements made no additional demands on the protocol, measurement time or hardware. Applying this approach to 18 volunteered 3D MRSI sets of 480 voxels each yielded N‐acetylaspartate, creatine, choline and myo‐inositol C_i^GM concentrations of 8.5 ± 0.7, 6.9 ± 0.6, 1.2 ± 0.2, 5.3 ± 0.6mM, respectively, and C_i^WM concentrations of 7.7 ± 0.6, 4.9 ± 0.5, 1.4 ± 0.1 and 4.4 ± 0.6mM, respectively. We showed that unaccounted voxel WM or GM partial volume can vary absolute quantification by 5–10% (more for ratios), which can often double the sample size required to establish statistical significance. Copyright © 2012 John Wiley & Sons, Ltd.     

Subcutaneous Tissue Mechanical Behavior is Linear and Viscoelastic Under Uniaxial Tension

Subcutaneous tissue is part of a bodywide network of “loose” connective tissue including interstitial connective tissues separating muscles and surrounding all nerves and blood vessels. Despite its ubiquitous presence in the body and its potential importance in a variety of therapies utilizing mechanical stretch, as well as normal movement and exercise, very little is known about loose connective tissue's biomechanical behavior. This study aimed to determine elastic and viscoelastic mechanical properties of ex-vivo rat subcutaneous tissue in uniaxial tension with incremental stress relaxation experiments. The elastic response of the tissue was linear, with instantaneous and equilibrium tensile moduli of 4.77 kPa and 2.75 kPa, respectively. Using a 5 parameter Maxwell solid model, material parameters μ₁ = 0.95 ± 0.24 Ns/m and μ₂ = 8.49 ± 2.42 Ns/m defined coefficients of viscosity related to time constants τ_1M = 3.83 ± 0.15 sec and τ_2M = 30.15 ± 3.16 sec, respectively. Using a continuous relaxation function, parameters C = 0.25 ± 0.12, τ_1C = 1.86 ± 0.34 sec, and τ_2C = 110.40 ± 25.59 sec defined the magnitude and frequency limits of the relaxation spectrum. This study provides baseline information for the stress-strain behaviors of subcutaneous connective tissue. Our results underscore the differences in mechanical behaviors between loose and high-load bearing connective tissues and suggest that loose connective tissues may function to transmit mechanical signals to and from the abundant fibroblasts, immune, vascular, and neural cells present within these tissues.     

Regions located in both the N-lobe and C-lobe of human lactoferrin participate in the binding interaction with bacterial lactoferrin receptors

As a first step in localizing the regions of human lactoferrin involved in binding to bacterial lactoferrin receptors, N-lobe and C-lobe fragments were assessed for binding to receptors on Neisseria meningitidis, Neisseria gonorrhoeae and Moraxella (Branhamella) catarrhalis. Preparations of N-lobe and C-lobe were obtained by tryptic digestion of iron-loaded human lactoferrin followed by separation of the two lobes by gel exclusion chromatography in 10% acetic acid. Solid phase binding studies demonstrated that the isolated C- and N-lobe preparations were capable of binding to membranes from iron-deficient N. meningitidis, N. gonorrhoeae and M. catarrhalis. The binding of the individual C- and N-lobes was confirmed by an analytical SDS-PAGE binding method in which the membrane-associated polypeptides were identified by prior biotinylation and subsequent binding of labelled streptavidin. This contrasts with bacterial transferrin receptors, which only bind to C-lobe fragment of human transferrin, indicating that the bacterial lactoferrin and transferrin receptors differ in their interaction with their respective glycoprotein ligands and may differ in the mechanism of iron removal.