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D Legrand

Publications and source records attributed to D Legrand.

At least 19 recordsLinked to original sources
PubMed2001

The binding of lactoferrin to glycosaminoglycans on enterocyte-like HT29-18-C1 cells is mediated through basic residues located in the N-terminus.

Although lactoferrins (Lfs) isolated from milk of various mammals exhibit a close structural relationship, they show species-specific binding to cells. To define the specificity of recognition of human (hLf), bovine (bLf) and murine (mLf) lactoferrin by human intestinal cells, we analysed the binding of the three proteins to a subclone derived from human carcinoma cell line HT29. We observed that hLf and bLf interact with two types of binding sites (K(d): 63+/-22 nM; 0.7+/-0.2 microM) while mLf was recognized only by the lowest affinity binding sites with a lower number of binding sites. Using N-terminal deleted human Lf variants, we found that the sequence G(1)RRRR(5) is mainly responsible for the interactions with HT29 cells. Lactoferrin-binding sites on the surface of HT29 cells were further identified as heparan sulphate and chondroitin sulphate glycosaminoglycans. We conclude that the presence of the sequence A(1)PRK(4) in bLf and K(1)ATT(4) in mLf provides an insight into why the interaction of bLf with cell membrane-associated glycosaminoglycans is similar to that of hLf and why binding of these lactoferrin species differs from that of murine Lf.

Amino Acid Sequence↗
PubMed2001

[Immune safety in blood transfusions].

Immune security of blood donation is defined by all means aiming at reducing or eliminating the immune risk related to blood donation. It concerns the whole transfusion process from the blood donor to the receiver. The immune risk of blood donation is directly related to the polymorphism of molecular and cellular blood groups systems. Immune security consists in avoiding the meeting of antigens and augmented by the direct baneful consequences of the immune conflict. This requires the previous immune characterisation of blood products and of the patients and of their compatibility, which must be strongly maintained along the transfusion process. To control this process, which is still too much deficient, represents the true guarantee of immune security of blood donation.

Blood Donors↗
PubMed2000

Lactoferrin inhibits the binding of lipopolysaccharides to L-selectin and subsequent production of reactive oxygen species by neutrophils.

The activation of leukocytes by lipopolysaccharides (LPS), resulting in the oxidative burst, contributes to the pathogenesis of septic shock. The binding of LPS to L-selectin, which was reported as a serum-independent LPS receptor on neutrophils, induces the production of oxygen free radicals. Human lactoferrin (hLf), an anti-inflammatory glycoprotein released from neutrophil granules during infection, binds to LPS. In this study, we investigated the capacity of hLf to inhibit the L-selectin-mediated activation of neutrophils. Our experiments revealed that hLf prevents the binding of LPS to L-selectin in a concentration-dependent manner. Inhibition was maximum (87.7+/-0.5%) at a concentration of 50 microg/ml of hLf. Furthermore, hLf inhibited up to 55.4+/-0.5% of the intracellular hydrogen peroxide production induced by LPS in neutrophils. These findings suggest that the anti-inflammatory properties of hLf are due, at least in part, to their ability to prevent the binding of LPS to neutrophil L-selectin.

Escherichia coli↗
PubMed2000

Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex.

Lipopolysaccharides (LPS), either in the free form or complexed to CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin (Lf), a LPS-chelating glycoprotein, protects animals against septic shock. Since optimal protection requires administration of Lf prior to lethal doses of LPS, we hypothesized that interactions between Lf and soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf (hLf) were used to determine the kinetic binding parameters of hLf to free sCD14 in an optical biosensor. The results demonstrated that hLf bound specifically and with a high affinity (K(d) = 16+/-7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A-2-keto-3-deoxyoctonic acid-heptose. In a second step, we have investigated whether the capacity of hLf to interact with sCD14 could modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by the sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC). Our experiments show that hLf significantly inhibited both E-selectin and ICAM-1 expressions at the surface of HUVEC. In conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to chelate LPS but also to its ability to interact with sCD14 and with the sCD14 complexed to LPS, thus modifying the activation of endothelial cells.

Amino Acid Sequence↗
PubMed1999

Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects.

The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane.

Amino Acid Sequence↗
PubMed1999

Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process.

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.

Animals↗
PubMed1998

Production of human lactoferrin in transgenic tobacco plants.

Production and characterization of human lactoferrin (hLf) in transgenic tobacco is reported. We have engineered two constructs containing either the native signal peptide from human lactoferrin or the signal peptide from sweet potato sporamin fused to human lactoferrin encoding cDNA. N-terminal sequences of rhLf purified from tobacco were identical to Lf from human milk for both constructs. The tobacco rhLf presents a molecular mass closely identical to native protein. Overall sugar composition shows the presence of plant specific xylose while sialic acid is absent. Binding parameters of the recombinant molecule to both Jurkat lymphoblastic T-cells or HT29-18-C1 enterocytes are similar to those of human lactoferrin isolated from milk.

Amino Acid Sequence↗
PubMed1998

Role of the first N-terminal basic cluster of human lactoferrin (R2R3R4R5) in the interactions with the Jurkat human lymphoblastic T-cells.

We previously characterized a receptor of Mr 105,000 for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of R2R3R4R5 of hLf in the interaction with cells, we studied the binding of hLf variants obtained either by tryptic proteolysis (hLf-2N, hLF-3N and hLf-4N) or by mutagenesis (rhLf-5N). Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. The binding parameters of bovine Lf and native hLf did not differ, whereas the binding parameters of murine Lf resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulfation, reduced the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N indicating that the hLf binding sites include sulfated molecules. The results suggest that the interaction of hLf with about 80,000 binding sites per Jurkat cell, mainly sulfated molecules, is dependent on R2R3R4, but not on R5. Interaction with about 20,000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. We conclude that the deletion of R2-R5 from hLf may serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.

Amino Acid Sequence↗
PubMed1998

Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein.

Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding of Escherichia coli 055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, and Limulus anti-LPS factor.

Acute-Phase Proteins↗
PubMed1998

[Pharmaco-economics: a point of view of the mutualities].

In Belgium, the mutualities are responsible for their budgets. They are playing an important role in drug pricing and reimbursement procedures. This document aims at explaining our line of arguments for a critical evaluation and the place for a pharmacoeconomic evaluation in the Belgian social security system. The available economic data for each molecule or pathology are essential but insufficient. Economic evaluations could help to reveal the costs and benefits of different uses of medical therapies and could thereby enhance the resources allocation in a health care system. But this science is still in its infancy, and in practice, the methodological quality of this kind of study is often weak. Furthermore it is not easy to transfer those evaluations to the Belgian reimburse systems. The clinical efficacy and the transparency is price setting should remain the two most important criterions to build reimbursement rules in order to ensure right price, rational use of medicine and accessibility for all.

Belgium↗
PubMed1997

The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells.

We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg2-Arg3-Arg4-Arg5 of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf-2N, hLf-3N and hLf-4N) from native hLf that had been treated with trypsin. Native hLf bound to 102000 sites on Jurkat cells with a dissociation constant (Kd) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf-5N) bound to 17000 sites with a Kd of 12 nM. The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; Kd 30 nM) resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N, indicating that the hLf-binding sites include sulphated molecules. We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg2-Arg3-Arg4, but not on Arg5. Interaction with approx. 20000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. Moreover, the affinity of hLf for the latter binding site is enhanced approx. 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.

Amino Acid Sequence↗
PubMed1997

Characterization of human lactoferrin produced in the baculovirus expression system.

Lactoferrin, an iron-binding 80-kDa glycoprotein, is a major component of human milk whose structure is now well defined. The binding site of lactoferrin to the membrane receptor of lymphocyte has been located in the region 4-52, but the amino acids directly involved in the interaction have not been identified yet. To gain further insights into the structure-function relationships of the lactoferrin binding site, we first expressed the cDNA encoding human lactoferrin in the lepidoptera Spodoptera frugiperda cells (Sf9) using a recombinant baculovirus. The selected transformant secreted and N-glycosylated protein of 78 kDa which was immunoprecipitated by specific anti-lactoferrin antibodies. To confirm the structure and the function of the recombinant lactoferrin, the protein was purified by ion-exchange chromatography and its physical, biochemical, and biological properties were compared with those of the native protein. In particular, the N-terminal amino acid sequence and the iron-binding stability as a function of pH, of both proteins, were identical. The main difference concerns the glycosylation which leads to glycans of lower molecular masses as detected by the electrophoretic mobility of lactoferrin after N-glycosidase F treatment and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Despite the different glycosylation features, the recombinant lactoferrin retained the binding property to the Jurkat human lymphoblastic T-cell line of the native lactoferrin. On the basis of these analyses, production of protein mutants generated by site-directed mutagenesis is now in process.

Animals↗
PubMed1996

Internalization of human lactoferrin by the Jurkat human lymphoblastic T-cell line.

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.

Cell Line↗
PubMed1995

Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide.

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

Amino Acid Sequence↗
PubMed1995

Structural determination of two N-linked glycans isolated from recombinant human lactoferrin expressed in BHK cells.

A full-length cDNA coding for human lactoferrin was isolated from a mammary gland library and the recombinant protein was expressed in BHK cells as described by Stowell K. M. et al. [1991, Biochem. J. 276, 349-355]. Two N-linked glycans from purified recombinant lactoferrin were released by hydrazinolysis and analyzed by 400-MHz 1H-NMR spectroscopy. The identified structures corresponded to N-acetyllactosaminic biantennary glycans and were alpha-2,3-disialylated forms (80%) or alpha-2,3-monosialylated (20%) forms. Moreover, 70% of total glycans were alpha-1,6-fucosylated at the GlcNAc residue linked to asparagine. In regard to its glycan moiety, the recombinant glycoprotein is close to native lactoferrins from milk or leucocytes but shows specific structural features which should be taken into account prior to in vivo and in vitro biological studies.

Animals↗