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Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins., (Copyright 1999 Wiley-Liss, Inc.)

This paper is concerned with the practical implementation of Monte Carlo simulation methods for charged particle transport. The emphasis is on light particles (electrons and positrons) because of the larger scattering and energy straggling effects. Differential cross sections (DCS) for the various interaction mechanisms are described. As the average number of interactions along the particle track increases with the initial energy, detailed simulation becomes unfeasible at high energies. We can then rely on mixed simulation algorithms: hard events (i.e. individual interactions with angular deflection or energy loss larger than given cutoff values) are sampled from the DCS whereas soft events are simulated by means of a multiple scattering approach. Too frequently, the statistical uncertainty of analogue simulation (i.e. strict simulation of the physical interaction process) is found to be so large that results are meaningless. This problem can be partially solved by applying simple variance reduction techniques.

Lactoferrin is an iron-binding glycoprotein found in exocrine secretions of mammals and released from neutrophilic granules during inflammation. This review describes the biological roles of lactoferrin in host defence. Secreted lactoferrin exerts antimicrobial action either by chelation of iron or by destabilization of bacterial membranes. Furthermore, lactoferrin modulates the inflammatory process, mainly by preventing the release of cytokines from monocytes and by regulating the proliferation and differentiation of immune cells. Some of these activities are related to the ability of lactoferrin to bind lipopolysaccharides (LPS) with high affinity. Indeed, recent in vitro studies indicate that lactoferrin is able to compete with the LPS-binding protein for LPS binding and therefore to prevent the transfer of LPS to CD14 present at the surface of monocytes. Moreover, the prophylactic properties of lactoferrin against septicemia in vivo have been demonstrated. Taken as a whole, these observations strongly suggest that lactoferrin is one of the key molecules which modulate the inflammatory response.

The aim of this study was to investigate effects of human lactoferrin (hLF) with regard to LFA-1 expression on unstimulated and lipopolysaccharide (LPS)-activated human peripheral blood mononuclear cells (PBMC). The investigations were carried out on 30 healthy volunteers, males and females, 24-58 years old. We found that hLF, at an optimal dose of 5 microg/ml/10(6) cells in 24-hour culture, exerted regulatory effects on LFA-1 expression, depending on distribution of this molecule on cells in control cultures and on the effects of LPS. First, we revealed several patterns of LFA-1 distribution and density of this marker among studied individuals. The effects of LPS and hLF on LFA-1 expression patterns were differential. LFA-1 expression was stimulated by individual actions of LPS or hLF, additive or synergistic effects of both factors, it could be also inhibited by hLF alone or in combination with LPS. In about one third of cases no significant effects of LPS or hLF on LFA-1 expression were seen. Removal of monocytes from the PBMC population diminished LFA-1 expression in control cultures and abolished LPS- or hLF-elicited changes. The regulatory effects of hLF were also blocked by treatment of PBMC cultures with anti-tumor necrosis factor alpha (TNF-alpha) antibodies. Taken together, the data showed that hLF and LPS had immunoregulatory properties with respect to LFA-1 expression on human PBMC and that these actions were mediated by monocytes and TNF-alpha.

We previously demonstrated that lactoferrin increases breast cell sensitivity to natural killer cell cytotoxicity whereas haematopoietic cells are unaffected by lactoferrin. It has been described that lactoferrin binds to various glycosaminoglycans. Compared to haematopoietic cells, breast cancer cells and particularly the breast cell line MDA-MB-231, possess a high level of proteoglycans. Scatchard analysis of 125I-lactoferrin binding to MDA-MB-231 cells revealed the presence of two classes of binding sites: a low affinity site with a Kd of about 700 nM and 3.9 x 10(6) sites and a higher affinity class with a Kd of 45 nM and 2.9 x 10(5) sites per cell. To investigate the potential regulation of lactoferrin activity by proteoglycans expressed on the MDA-MB-231 cells, we treated these cells with glycosaminoglycan-degrading enzymes or sodium chlorate, a metabolic inhibitor of proteoglycan sulphation. We showed that chondroitinase treatment has no effect, while heparinase or chlorate treatment significantly reduces both the binding of lactoferrin to cell surface sulphated molecules such as heparan sulphate proteoglycans (HSPG) and the affinity of lactoferrin for the higher affinity binding sites. The modulation of the lactoferrin binding was correlated with a decrease in lactoferrin activities on both MDA-MB-231 cell sensitisation to lysis and proliferation. Taken together, these results suggest that the presence of adequately sulphated molecules, in particular HSPG, is important for lactoferrin interaction and activity on the breast cancer cells MDA-MB-231.

Lactoferrin is an iron-binding glycoprotein implicated in particular in the control of immune functions and cell proliferation. We have investigated its involvement, at inflammatory concentrations, in cancer progression. We report that lactoferrin has a significant effect on natural killer (NK) cell cytotoxicity against haematopoietic and breast epithelial cell lines. Lactoferrin increases cytolysis at a low concentration (10 micrograms/ml) while at a high concentration (100 micrograms/ml) it modulates cytolysis depending on the target cell phenotype. By pre-treatment of either NK cells or target cells with lactoferrin, we have demonstrated that the lactoferrin effect is due both to a modulation of NK cell cytotoxicity and the target cell sensitivity to lysis. Lactoferrin binds to 91% of the naturally heterogeneous CD56dim/bright NK cell population and increases the NK cell cytotoxic activity at low concentrations. High concentrations of lactoferrin seem to be toxic for the CD56bright NK cells and decrease NK cell cytotoxicity. Lactoferrin also exerts an effect on target cells depending on the cell phenotype. It does not modify the susceptibility to lysis of haematopoietic cells such as Jurkat and K-562 cells, but does significantly increase that of the breast and colon epithelial cells. We have also demonstrated that lactoferrin inhibits epithelial cell proliferation by blocking the cell cycle progression.

The aim of this study was to monitor several immune parameters in 17 healthy volunteers taking orally commercially available capsules containing bovine lactoferrin (BLFT) for 10 days (40 mg of BLFT daily). We determined leukocyte number and content of main blood cell types, spontaneous and phytohemagglutinin A (PHA)-induced proliferation of lymphocytes, plasma levels of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) as well as spontaneous and lipopolysaccharide (LPS)-induced production of these cytokines in peripheral blood cell cultures. All measurements were performed before, one day and 14 days following cessation of BLFT treatment. We established some, transient drop in the percentage of neutrophils accompanied by an opposite phenomenon with regard to lymphocyte levels. More profound changes were registered in the percentage of other cell types, for example a 100% increase in the level of immature cell forms (bands) was noted. At the same time the percentages of eosinophils and monocytes declined significantly. All these changes were, however, more individual and regulatory, the direction of these changes depended on initial picture of blood cells. Although the proliferative response of lymphocytes showed, on average, a transient decrease, differentiated effects of BLFT treatment were observed depending on initial ability of lymphocytes to proliferate. TNF-alpha serum levels showed a tendency to decrease during the monitoring time, the changes of IL-6 levels were, however, not significant. As in the case of the proliferative response, the treatment with BLFT was regulatory with respect to serum TNF-alpha levels. When we analyzed spontaneous and LPS-induced cytokine production in cell cultures we found that mainly the mean spontaneous response was affected (inhibition). We also observed a typical, regulatory action of BLFT on the level of spontaneously produced IL-6. This kind of regulatory action of BLFT was also found in the case of spontaneously produced TNF-alpha in cell cultures. The influence of other ingredients such as selenium or vitamins, contained in the capsules, cannot be excluded, although our latest data showed that orally taken bovine lactoferrin alone can induce identical changes as BLFT. Taken together, we revealed regulatory effects of oral treatment with commercially available capsules, containing BLFT. The results indicate that oral administration on BLFT-containing capsules may regulate some immune parameters in healthy individuals. In addition, the data suggest that bovine lactoferrin may be applied in clinic to improve the immune status of patients.

A rapid, sensitive, and reproducible flow cytofluorimetric procedure is described for quantitation of erythrophagocytosis based on the use of red blood cells (RBCs) labeled with the fluorescent probe PKH-26. The procedure involves the following steps: i) incubation of PKH-26-labeled erythrocytes with macrophages, ii) removal of un-bound red blood cells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macrophages. Each step was controlled by fluorescence microscopy. Use of fluorescent, instead of radio-labeled RBCs, makes the method more sensitive, rapid, and avoids radioactive hazards. Furthermore, this approach is multi-parametric and can distinguish different populations of macrophages with reference to their erythrophagocytic potential. This technology moreover, has broad applications from the initial step of contact (between effector and target cells to study the specific receptor mediated attachment) to the subsequent cascade of time-dependent changes resulting from that signal transduction.

The effect of human lactoferrin on the human lymphoblastic T cell line (Jurkat) was tested with regard to proliferation and differentiation. Lactoferrin enhanced cell proliferation in a serum-reduced (1% fetal calf serum) culture. The stimulatory effect of lactoferrin on proliferation depended on the degree of iron saturation but the amplitude of the effect was low, similar to that obtained in the presence of serum transferrin. The proliferation stimulatory effect of lactoferrin was not observed in the presence of 10% fetal calf serum (FCS) in the culture medium. These results suggest that Fe-lactoferrin can substitute for Fe-transferrin during the prolonged culture of cells in a low serum concentration. Iron-saturated lactoferrin was also shown to promote T cell differentiation. Jurkat cells, when exposed to iron-saturated lactoferrin in the presence of 10% FCS, gradually exhibited a decrease in the cell volume, cell surface density of CD71 antigen, the nuclear incorporation of [methyl-3H]thymidine, but an increase of the percentage of cell population in the G0/1 phase of the cell cycle. These modifications were accompanied by the appearance of CD4 antigen at the cell surface. Therefore, in the continuous presence of lactoferrin, proliferating cells slowly enter into quiescence state, undergoing cell differentiation.

Binding of either iron-saturated or iron-free lactoferrin to the Jurkat human lymphoblastic T-cell line was saturable with a dissociation constant Kd of 40 nM. The total number of binding sites was estimated to be approximately 300,000. Non-specific binding did not exceed 30% of the total binding. Removal of the 4 clustered arginine residues of lactoferrin at position 2 to 5, which are involved in the interactions with heparan sulfate, did not modify the binding parameters. Therefore, the high number of low affinity binding sites previously described as responsible for the interaction of lactoferrin with either hepatocytes, enterocytes or the U937 monocytic cell line, is not involved in the binding of lactoferrin to Jurkat cells. After binding at 4 degrees C, a shift to 37 degrees C causes cell to internalize lactoferrin, with the maximum intracellular concentration found at 3 to 8 and 5 to 15 min for iron-saturated and iron-free forms, respectively. Addition of colchicine had no effect on binding or internalization. These results suggest that endocytosis of lactoferrin by Jurkat cells occurs through a receptor-mediated process. Jurkat cells internalize lactoferrin monophasically with a first-order endocytic constant K(in) of 0.060 min-1 at 37 degrees C. Confocal microscopic analysis, using fluorescein-carbohydrate-labeled lactoferrin showed that lactoferrin was mainly localized in intracellular vesicles. Following uptake, the endocytic path utilized by fluorescein-carbohydrate-labeled lactoferrin was shown to diverge from that of rhodamine-labeled serum transferrin; after internalization, lactoferrin and serum transferrin did not fully colocalize. Intracellular lactoferrin was found in endosome vesicles as assessed by electron microscopy. Raising the pH in endosomes using chloroquine led to the accumulation of lactoferrin into endosomes (acidic compartment). After internalization, Jurkat cells released both degraded and intact lactoferrin into the culture medium, suggesting that a fraction (30-40%) of the ligand is degraded at each round of endocytosis.

Bovine (BLF) and human (HLF) lactoferrin inhibit proliferation and cytokine production by an antigen specific TH1 cell line stimulated with antigen presenting cells (APC) and antigen. Beside the inhibitory effects on TH1 cells we observed a decrease of interleukin 2 receptor (IL-2R) expression on these cells contacted with LF. Lactoferrin (LF) did not inhibit proliferation of indicator HT-2 cells to IL-2 suggesting lack of interference with IL-2/IL-2R interaction by LF. No inhibitory effect was observed on proliferation and cytokine production by TH2 cell lines and no changes in interleukin 4 receptor (IL-4R) levels were found with regard to TH2 cell line. We conclude that LF has differential activity with regard to effector functions of antigen-specific T cells by inhibiting TH1 but not TH2-mediated immune responses.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

The results presented in this communication show that New Zealand Black (NZB) mice treated for a prolonged period with bovine lactoferrin (BLF) exhibit a decreased frequency of positive Coombs' reaction. This effect was dose dependent and best pronounced at a dose of 50 micrograms/dose. We found, in addition, that incubation of peritoneal cells with BLF resulted in a decreased number of cells recognizing Hb antigen on autologous erythrocytes. The data indicated that lactoferrin may be of therapeutical value in treatment of autoimmune disorders.

Monoclonal antibodies have been raised against the soluble lactotransferrin binding protein purified from the cell culture supernatant of Jurkat cell line, a human T-lymphoblastic cell. All monoclonal antibodies were able to specifically bind to the membrane of Jurkat cells. One of the monoclonal antibodies, DP5B3G10, recognized both the soluble lactotransferrin-binding protein and the membrane lymphocyte lactotransferrin receptor after SDS-PAGE in presence of 2-mercaptoethanol and electrotransfer on nitrocellulose. The monoclonal antibody DP5B3G10 inhibited the binding of lactotransferrin to Jurkat cells and human peripheral activated lymphocytes. In addition, lactotransferrin inhibited the binding of the monoclonal antibody to the cell surface. These results suggest that the 95 kDa lactotransferrin-binding protein isolated from the cell culture medium corresponds to the soluble form of the 105 kDa lymphocyte lactotransferrin receptor. Corresponding proteins of 105 kDa molecular mass were identified in Jurkat and CEM T-cells and Raji B-cells. Finally, the monoclonal antibody DP5B3G10 was used to immunolocalize the lactotransferrin receptor on the Jurkat cells. Using fluorescence and electron microscopy, the receptor was localized both inside and at the cell surface. The cell membrane receptor was associated into clusters. After permeabilization of the plasma membrane, the staining was positive in the peri-membrane area. The region near the nucleus was devoid of receptor.

Background: Lectins mediate cell-cell interactions by specifically recognizing oligosaccharide chains. Legume lectins serve as mediators for the symbiotic interactions between plants and nitrogen-fixing microorganisms, an important process in the nitrogen cycle. Lectins from the Viciae tribe have a high affinity for the fucosylated biantennary N-acetyllactosamine-type glycans which are to be found in the majority of N-glycosylproteins. While the structures of several lectins complexed with incomplete oligosaccharides have been solved, no previous structure has included the complete glycoprotein., Results: We have determined the crystal structures of Lathyrus ochrus isolectin II complexed with the N2 monoglycosylated fragment of human lactotransferrin (18 kDa) and an isolated glycopeptide (2.1 kDa) fragment of human lactotransferrin (at 3.3 A and 2.8 A resolution, respectively). Comparison between the two structures showed that the protein part of the glycoprotein has little influence on either the stabilization of the complex or the sugar conformation. In both cases the oligosaccharide adopts the same extended conformation. Besides the essential mannose moiety of the monosaccharide-binding site, the fucose-1' of the core has a large surface of interaction with the lectin. This oligosaccharide conformation differs substantially from that seen in the previously determined isolectin I-octasaccharide complex. Comparison of our structure with that of concanavalin A (ConA) suggests that the ConA binding site cannot accommodate this fucose., Conclusions: Our results explain the observation that Viciae lectins have a higher affinity for fucosylated oligosaccharides than for unfucosylated ones, whereas the affinity of ConA for these types of oligosaccharides is similar. This explanation is testable by mutagenesis experiments. Our structure shows a large complementary surface area between the oligosaccharide and the lectin, in contrast with the recently determined structure of a complex between the carbohydrate recognition domain of a C-type mammalian lectin and an oligomannoside, where only the non-reducing terminal mannose residue interacts with the lectin.

Lactotransferrin (Lf), an iron-binding glycoprotein present as a major component in the specific granules of human neutrophilic granulocytes is released in the blood during the acute phase of infection and participates in the regulation of the host-defence mechanisms. Our previous observations (Mazurier et al., 1989) showing i) that the activation by PHA of T-lymphocytes induces the appearance at the cell surface of Lf-receptors which are absent from the membrane of resting lymphocytes and ii) that Lf becomes a growth factor for the activated lymphocytes, led us to undertake a series of researches on the presence of Lf receptors at the surface of different blood cells. Characterization of Lf receptors was performed by flow cytofluorimetry using either Lf labelled on its glycan moiety with fluorescein or purified anti-lymphocyte Lf receptor antibodies. High affinity receptors for Lf were characterized only at the surface of human activated lymphocytes and of non-activated platelets. These two receptors possess common physicochemical properties and antigenic epitopes. Low affinity receptors for Lf were characterized on monocytes, eosinophils and neutrophils. These receptors are immunologically different from those found on activated lymphocytes and on non-activated platelets. Cell-lines of human lymphocyte T and megakaryocyte possess lactotransferrin receptors whose properties are similar to those found on peripheral blood cells. The soluble form of the receptor identified in the lymphocytes T culture medium possesses a molecular mass close to that of the membrane receptor suggesting that the cytoplasmic tail of the receptor should be very short.

In order to establish relationships between glycan structure and biological activity, the authors undertook a comparative study of the glycan primary structure of different transferrins from several species. By associating permethylation-mass spectrometry and 1H-NMR spectroscopy, the primary structure of the human, bovine, caprine, murine and porcine lactotransferrin glycans were determined. Using the same methods, the glycan structure of 9 serotransferrins was determined. The results obtained led to the conclusion that glycans are specific for each transferrin and, for a given transferrin, specific to the species. No relationship could be established between primary structure and function of transferrin glycans. Glycan molecular modelling, molecular dynamics simulations and X-ray diffraction studies of free glycans confirm the mobility in space of antennae. In contrast, the glycan associated with a protein is immobilized into only one conformation, as in the case of glycan-lectin associations or of "internal" glycan-protein interactions, like in rabbit serotransferrin, in which the glycan forms a bridge between the two lobes of the peptide chain, and maintains the protein in a biologically active conformation. In the case of human sero- and lactotransferrins, the glycans are in an external position on the molecules and could play a role of recognition signals.

A fluorescent lactotransferrin probe was prepared by coupling 5-(([2-(carbhydrazino)methyl]-thio)acetyl)amino fluorescein to aldehyde groups that were produced by a mild periodic-acid oxidation of the glycan moieties of lactotransferrin. In this manner, the receptor-binding site of the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein isothiocyanate. The fluorescent probe allowed us to characterize, by flow cytometry, the binding of lactotransferrin to non-activated human platelets. The putative lactotransferrin platelet receptor was purified and its immunological and physico-chemical properties were found to be very similar to those of the receptor previously isolated from activated human lymphocytes. Lactotransferrin inhibits ADP-induced platelet aggregation at concentrations down to 5 nM, which can be reached in the plasma after leukocyte degranulation. Inhibition of platelet aggregation was also observed with the N-terminal fragment of lactotransferrin (residues 3-281; 50% inhibition = 2 microM) and with CFQWQRNMRKVRGPPVSC synthetic octodecapeptide (residues 20-37; 50% inhibition = 20 microM) corresponding to one of the two external loops (residues 28-34 and 39-42) where we recently located the receptor-binding site. The activity (50% inhibition = 500 microM) of the tetrapeptide KRDS (residues 39-42), which has already been described, was at least 25-times and 16000-times lower than the activity of the octodecapeptide and of the lactotransferrin molecules, respectively. Finally, the inhibition was demonstrated to be mediated by a mechanism which requires the binding of lactotransferrin to its putative receptor and not to platelet glycoprotein IIb-IIIa.

Isolectin II (LOL II) isolated from the seeds of Lathyrus ochrus has been crystallized in the presence of the N2 fragment (18,500 Da) isolated from human lactotransferrin, which contains an N-acetyllactosamine type biantennary glycan linked to Asn137. This is the first example of a legume lectin crystallized with an N-glycosylprotein. Crystals of the LOL II-N2 complex belong to the tetragonal space group (P4(1)2(1)2 or the enantiomorph) with cell dimensions: a = b = 63.5 A, c = 251.9 A. They diffract well up to at least 3.5 A resolution and more weakly up to 2.8 A resolution. Assuming one functional half-entity in the asymmetric unit, an alpha, beta monomer complexed to one N2 fragment (24,500 Da + 18,500 Da) would give a Vm of 2.95 A3/Da and a solvent content of approximately 58%. SDS/polyacrylamide gels of the dissolved crystals show the presence of both the LOL II and N2 fragment.

Fluorescein isothiocyanate derivatization of the human lactotransferrin on Lys-264 inhibits the binding of the protein of human PHA-activated lymphocytes [Legrand, D., Mazurier, J., Maes, P., Rochard, E., Montreuil, J., & Spik, G. (1991) Biochem. J. 276, 733-738], indicating that part of the receptor-binding site is located in the N-terminal domain I of lactotransferrin. In the present study, a 6-kDa peptide (residues 4-52) was isolated from the N-terminal lobe of human lactotransferrin which inhibited the binding of the protein to its cell receptor. In addition, lactotransferrin was derivatized using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and sulfosuccinimidyl 6-((4'-azido-2'-nitrophenyl)amino)hexanoate (sulfo-SANPAH), two heterobifunctional reagents generally used for receptor-ligand cross-linking. The azide group of these two reagents was inactivated by photolysis, and only the succinimidyl ester group was allowed to react with lysine residues of the protein. The binding of the derivatized lactotransferrins to the human lymphocyte receptor was assayed. SASD, which binds to Lys-74, was able to inhibit the binding of lactotransferrin to the cell receptor, in contrast to Lys-281-binding sulfo-SANPAH. Molecular modeling showed the position of SASD, sulfo-SANPAH, and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask residues 4-6 and two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). The comparison of the primary and tertiary structures of human lactotransferrin and serotransferrin, which bind to specific cell receptors, shows that the above-mentioned regions, which are likely involved in protein-receptor interactions, possess specific structural features.

The conformations of a disialylated monofucosylated biantennary glycan of the N-acetyllactosamine type were analysed using the Tripos 5.3 force field from the Sybyl software currently used for molecular modelling. The conformation of each glycosidic linkage was calculated when included in oligosaccharide structures of up to 5 units and the influence of the glycosidic environment on the overall structure was measured. The study clearly shows that the conformation of a branched glycan cannot result from the simple addition of the different low energy conformers of each of the glycosidic linkages constituting the glycan structure. The asymmetrical conformation of the two antennae was demonstrated. The lowest energy conformations of the overall glycan structure were built and classified into 5 main models: the Y, T, bird and broken wing conformations already described and a new one called the 'back folded wing conformation.'

Human milk lactotransferrin at a concentration ranging from 1 to 10 micrograms/ml stimulated up to 5 times the humoral immune response to sheep red blood cells, expressed as the number of plaque-forming cells, when injected into mice 3 h before immunization. Further, lactotransferrin-treated thymocytes given intravenously into mice, enhanced the immune response to sheep red blood cells to the same extent as IL-1. In vitro, studies showed that CD4- CD8- thymocytes incubated with lactotransferrin and added to the splenocyte cultures, increased the immune response to sheep red blood cells. Flow cytometry analysis studies indicated that, after an overnight incubation with human lactotransferrin, CD4- CD8- thymocytes acquired the CD4 antigen characteristic for the helper cell phenotype. Taken together, these results suggest that lactotransferrin stimulates the immune response by a process which involves the promotion of T cell differentiation.

On the basis of experimental data and of computer calculations using the Tripos 5.3 force field in order to examine the three-dimensional structures which are sterically feasible and the conformations which are energetically the most favourable, we have designed a program of molecular modelling of biantennary glycans of the N-acetyllactosaminic type (complex type). We demonstrate that, in absence of any interaction with the protein, a high number of glycan conformations exists which can be classified into five basic conformations, four of which have already been described. In fact, in addition to the Y-, T-, "bird" and "broken-wing" conformations, a "back-folded wing" conformation is energetically feasible. In contrast, the glycan linked to the protein is immobilized into only one conformation: the "broken-wing" conformation. Forming a bridge between the two lobes of the peptide chain, it probably contributes to the maintenance of the protein in a biologically active conformation.

The recent determination by X-ray diffraction of the tridimensional structure of human lactotransferrin has underlined the presence of two lobes, each composed of two domains, I and II, as well as the involvement of five ligands in the binding of iron. Only one of the ligands (Asp-61) is located in domain I (residues 1-90 and 252-320), while the others [two tyrosine, one histidine and one (bi)carbonate ion linked to an arginine residue] belong to domain II (residues 91-251). On the basis of these data and of our previous results concerning the isolation of the 30 kDa N-tryptic fragment (residues 4-281) and the 20 kDa N2-glycopeptide (N-terminal domain II; residues 91-253) from human and bovine lactotransferrins, we have compared the iron-binding properties of these two fragments. The results demonstrate that Asp-61, which is missing from domain II, does not take part in the stability upon protonation of the iron complex of both human and bovine lactotransferrins. Furthermore, by comparing the iron-binding properties of human and bovine lactotransferrins to those of isolated 30 kDa N-tryptic and 50 kDa C-tryptic fragments and of the reassociated N,C-tryptic complex of both proteins, it has been shown that the non-covalent interactions which occurred between the two lobes of lactotransferrins and in the reassociated N,C-tryptic complex can explain in part the high affinity of lactotransferrins for iron. Finally, deglycosylation experiments on the 30 kDa N-tryptic fragment and N-terminal domain II from human and bovine lactotransferrins demonstrate that full removal of the glycan moiety leads to the loss of iron-binding capacity and so underlines the importance of the glycan moiety in the stability upon protonation of the N-terminal iron-binding site of both lactotransferrins.

Several lines of evidence have recently suggested the occurrence of a specific lactotransferrin receptor in the small intestinal brush-border membrane in several animal species, which is thought to be involved in lactotransferrin-mediated intestinal iron absorption. We report here for the first time the isolation and partial characterization of this receptor from mouse intestinal brush border. The receptor has been purified to homogeneity by affinity chromatography on an immobilized human lactotransferrin column. The purified receptor was found to be active in that it binds iron-free and iron-saturated lactotransferrin with a Kd of 0.1 microM. Anti-receptor antibodies were prepared, and the receptor was further isolated by immunoaffinity chromatography in higher yield but in a denatured form. The purified receptor was revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis to be a protein of about Mr = 130,000, consisting of a single polypeptide chain. The isoelectric point was determined to be 5.8. The receptor was further shown to bear concanavalin A and phytohemagglutinin L binding glycans. Digestion by N-glycanase and endo-N-acetyl-beta-D-glucosaminidase B led to a decrease of Mr = 25,000, while the endo-N-acetyl-beta-D-glucosaminidase H was uneffective, suggesting that the lactotransferrin receptor is mainly glycosylated by bi- and triantennary glycans. To gain further insight into the interaction of the receptor with lactotransferrin, namely, the number of ligand molecules bound per molecule of receptor, mouse lactotransferrin was cross-linked to its membrane-bound enterocyte receptor by use of radiolabeled sulfosuccinimidyl 3-[[2-(p-azidosalicylamido)ethyl]dithio]propionate (SASD).(ABSTRACT TRUNCATED AT 250 WORDS)

Mild treatment of iron-saturated human lactotransferrin by trypsin at pH 8.2 cleaves the molecule into a N-tryptic (Mr approximately equal to 30000) and a C-tryptic (Mr approximately equal to 50000) fragment, which have been isolated. Each of them carries a glycan moiety and keeps the property to bind reversibly one Fe3+. The N-tryptic fragment has been submitted to a second tryptic digestion which led to an iron-binding glycopeptide fragment with a molecular weight of about 18500. This fragment, the smallest iron-binding peptide isolated up to now from a transferrin, includes the ND2 domain of human lactotransferrin.

In this review article, human lactotransferrin is compared to human serum transferrin and hen ovotransferrin. For the first time the possibility of a 6-fold internal homology of the transferrins is raised: a scheme in which 6 domains are defined is reported; two of them with the highest homology seem to be implicated in the 2 iron binding sites of each transferrin. The location of the disulfide bridges of the 3 transferrins and of their prosthetic sugar groups is discussed: some not yet described half-cystine containing lactotransferrin peptides are indicated.

The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120-128). In the present data, using 125I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 X 10(13) sites per mg of membrane proteins with an equilibrium constant of 1.2 X 10(6) M-1. Sodium dodecyl sulfate solubilization of the brush-border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with 125I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border.

The optimal conditions of the binding of human lactotransferrin to human alveolar macrophages have been determined and the necessity to measure the binding in absence of bovine serum albumin was demonstrated. In these conditions, diferric lactotransferrin and iron-free lactotransferrin are reversibly bound with the following parameters: association constant Ka = 2 and 5 X 10(6) M-1, respectively, and the number of binding sites N = 1.2 and 1 X 10(7), respectively. The binding of the two forms of lactotransferrins was inhibited by various neoglycoproteins, the highest inhibition being obtained with L-fucosyl, then, in the following decreasing order: D-mannosyl greater than N-acetyl-D-glucosaminyl greater than D-galactosyl. In the same conditions, the binding of serotransferrin (Ka = 2 X 10(7) M-1 and 1.6 X 10(7) M-1; N = 5 X 10(4) and 8 X 10(4) for diferric and iron-free protein, respectively) was not inhibited. These results suggest that the recognition of lactotransferrin is mediated by one or several membrane lectins, the fucose being one of the sugar playing an important role in the association. On the contrary, the binding of serotransferrin does not depend on a membrane lectin system.

The complete amino acid sequence (703 amino acid residues) of human lactotransferrin has been determined. The location of the disulfide bridges has also been investigated. Computer analysis established internal homology of the two domains (residues 1-338 and residues 339-703). Each domain contains a single iron-binding site and a single glycosylation site (asparagine residues 137 and 490) located in homologous positions. Prediction of the secondary structure of the two homologous moieties of human lactotransferrin has also been performed. The present results allowed a series of comparisons to be made with human serum transferrin and hen ovotransferrin.

The previously described temperature and pH-dependent transition in the solid state of hen lysozyme was studied in solution. Experiment concerning the velocity of lysis of M. luteus by lysozyme and its behavior in presence of an inhibitor (GlcNAc) as well as a reinvestigation of the Arrhenius curves over a large range of pH, demonstrated the existence of two temperature-induced domains. An inhibitor-insensitive lysozyme form was characterized at 40 degrees (physiological temperature).

Human lactotransferrin is able to bind two vanadyl(IV) ions in specific metal-binding sites. The EPR signals of the two vanadyl bound ions, however, appear as one. This result suggests that the environments of the binding sites of human lactotransferrin are similar. The binding activity is promoted to pH 4 using carbonate or bicarbonate as synergistic anion. This unusual stability of the anion-binding site, which is destroyed below pH 6 for other transferrins, can explain in part the great stability of the metallic complexes of human lactotransferrin. However, the different sensitivities of the two metal-binding sites towards protonation permit the preparation of mixed vanadyl(IV), iron(III) complexes with VO2+ bound either on the N-terminal (acid-labile or B site) or on the C-terminal (acid-stable or A site) site. Analysis of the spectra of such mixed complexes shows the presence of a third nonspecific VO2+-binding site termed A'. The nonspecific A' site seems to be located on the outer surface of the protein close to the C-terminal site.

Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96], has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 nM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).

In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time-dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen-stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum-free medium increased the proliferative activity of phytohemagglutinin-stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron-saturated lactotransferrin at a concentration of 0.17 microM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin. The lactotransferrin receptor was visualized using 125I-labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X-100 extract of the phytohemagglutinin-stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass. Purification of the lactotransferrin receptor from the Triton-X-100-soluble extract of stimulated lymphocytes was performed by antiligand-affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.

Transferrins are iron-binding glycoproteins involved in iron metabolism and antibacterial defense mechanisms. Since the discovery of transferrins, many studies have attempted to characterize the iron ligands and to establish the conformation of the iron-binding sites. From chemical and spectroscopic studies, it was generally accepted that iron was hexacoordinated to Tyr and His residues, to a water molecule and to a (bi)carbonate ion, electrostatically linked to an Arg residue. On the basis of these studies, on the one hand, and on the basis of the homologies between the amino acid sequences of transferrins, on the other hand, predicted data have been provided about the number and location of the iron ligands. Recent X-ray crystallography studies of human lactotransferrin have partially confirmed the above-mentioned predicted data and have brought invaluable information about the nature of the ligands and the conformation of the iron-binding site. On the basis of the obtained results, a scheme has been proposed in which the iron is coordinated to 2 Tyr, 1 His and 1 Asp residues, to a (bi)carbonate linked to an Arg residue and probably to a water molecule. The iron-binding site is located at the interface between the two domains which constitute each lobe of the transferrins.

Authors describe an original procedure to prepare pure lactotransferrin from cow milk. Physicochemical properties of this lactotransferrin have been studied and compared with results from others. New data are presented: presence of fucose and N-acetylgalactosamine; C-terminal amino-acid identified with threonine. On the other hand, 4 fractions have been obtained by "DEAE-Sephadex" chromatography, study of which demonstrates that the microheterogeneity of the lactotransferrin depends on the carbohydrate moiety and especially on the N-acetylneuraminic acid content which varies from 0 to 2 residues.

The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technique. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with 125I-labelled lactotransferrin and lactotransferrin dual-labelled with 59Fe and 125I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.

In order to determine the conformational relationship of iron binding of human serotransferrin and lactotransferrin, ultraviolet difference spectral studies were performed in the presence of guanidine chloride and perturbants as deuterium oxide, ethylene glycol, glycerol and polyethylene glycol. In the presence of guanidine chloride solution the molar absorption differences at 292 nm of iron-saturated forms versus iron-free forms of human serotransferrin and lactotransferrin are respectively -16000 +/- 1000 and -14000 +/- 775. These modifications may be attributed to the involvement of tryptophan residues in the iron-binding sites of the two proteins. However, the results do not demonstrate that these tryptophan residues are bound directly to iron. Difference spectral studies in the presence of perturbants show that the apparent exposed tryptophan and tyrosine residues are higher with shorter range perturbants in iron-free forms of both transferrin molecules. The most important modification of exposed tyrosine residues has been noticed upon removing iron from human lactotransferrin than from serotransferrin.