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At least 19 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed1996

Recognition of lactoferrin and aminopeptidase M-modified lactoferrin by the liver: involvement of proteoglycans and the remnant receptor.

1. Lactoferrin and aminopeptidase M-modified lactoferrin (APM-lactoferrin; which lacks its 14 N-terminal amino acids) inhibit the liver uptake of lipoprotein remnant. In the present study, the role of proteoglycans in the initial interaction of beta-migrating very-low-density lipoprotein (beta-VLDL), native and APM-lactoferrin with isolated rat parenchymal liver cells was investigated. Treatment of the cells with chondroitinase lowered the Kd of lactoferrin binding (from 10 to 2.4 microM), and the number of sites/cell (from 20 x 10(6) to 7 x 10(6)), while heparinase treatment did not affect the binding. The binding characteristics of APM-lactoferrin and beta-VLDL were not altered by treatment of the cells with chondroitinase or heparinase. It is concluded that proteoglycans are not involved in the initial binding of APM-lactoferrin and beta-VLDL to parenchymal cells, while chondroitin sulphate proteoglycans are mainly responsible for the massive, low-affinity binding of native lactoferrin..2. The binding of lactoferrin, APM-lactoferrin and beta-VLDL to parenchymal liver cells was not influenced by the glutathione S-transferase-receptor-associated protein (GST-RAP) (97.2% +/- 4.0%, 95.5 +/- 3.7% and 98.5% of the control binding), while the binding of alpha 2-macroglobulin was fully blocked at 10 micrograms/ml GST-RAP (1.8 +/- 0.5% of the control binding). Since GST-RAP blocks the binding of all the known ligands to the low-density lipoprotein (LDL)-receptor-related protein (LRP), it is concluded that LRP is not the initial primary recognition site for lactoferrin, APM-lactoferrin and beta-VLDL on parenchymal liver cells. 3. We showed earlier that.APM-lactoferrin, as compared with lactoferrin, is a more effective inhibitor of the liver uptake of lipoprotein remnants (49.4 +/- 4.0% versus 80.8 +/- 4.8% of the control at 500 micrograms/ml respectively). We found in the present study that beta-VLDL is able to inhibit the binding of APM-lactoferrin to parenchymal liver cells significantly (74.9 +/- 3.3% of the control; P < 0.002), while the lactoferrin binding was unaffected. It is concluded that a still unidentified specific recognition site (the putative remnant receptor) is responsible for the initial binding of remnants to parenchymal cells and it is suggested that the partial cross-competition between APM-lactoferrin and beta-VLDL may be of further help in the elucidation of the molecular nature of this recognition site.

Aminopeptidases↗
PubMed1996

Recognition of lactoferrin and aminopeptidase M-modified lactoferrin by the liver: involvement of the remnant receptor.

Lactoferrin inhibits the hepatic uptake of lipoprotein remnants, and we showed earlier that arginine residues of lactoferrin are involved. In this study, lactoferrin was treated with aminopeptidase-M (APM), which resulted in removal of 14 N-terminal amino acids, including 4 clustered arginines at positions 2-5 (APM-lactoferrin). After i.v. injection into rats, 125I-APM-lactoferrin was cleared within 10 min by the liver parenchymal cells (74.7% of the dose). Binding of APM-lactoferrin to isolated parenchymal liver cells was saturable with a Kd of 186 nM (750.000 sites/cell). This is in striking contrast to the binding of lactoferrin (Kd 10 microM; 20 x 10(6) sites/cell). Preinjection of rats with 20 mg of APM-lactoferrin/kg body weight reduced the liver association of beta-VLDL by 50%, whereas lactoferrin had no effect at this dose. With isolated parenchymal liver cells, APM-lactoferrin was a more effective competitor for beta-VLDL binding than native lactoferrin (50% inhibition at 0.5 mg/ml vs. 8.0 mg/ml). We conclude that the 4-arginine cluster of lactoferrin at position 2-5 involved in the massive association of lactoferrin with the parenchymal liver cell, but is not essential for the inhibition of the lipoprotein remnant uptake. The Arg/Lys sequence at position 25-30, which resembles the binding site of apoE, may mediate the high affinity binding of lactoferrin and block the binding of beta-VLDL to the remnant receptor efficiently.

Aminopeptidases↗
PubMed1995

Receptor-mediated binding of milk lactoferrin to nursing piglet enterocytes: a model for studies on absorption of lactoferrin-bound iron.

Lactoferrin, an iron-binding glycoprotein that is abundant in milk of some species, has been suggested to play a key role in the absorption of iron in human infants. This hypothesis is based on the dominant role of lactoferrin as an iron-binding component in human milk and on the occurrence of lactoferrin receptors in brush-border membranes in infants' intestines. The piglet may be a useful model to evaluate the biological activity of lactoferrin because we have previously found the presence of a lactoferrin receptor in brush-border membranes from suckling piglets. In this study, viable enterocytes were isolated from 6- to 20-day-old suckling piglets. Binding studies were performed at 4 degrees C using 125I-labeled porcine lactoferrin. Scatchard analysis of equilibrium binding data showed an apparent binding constant (Kd) of 2 x 10(-6) M (SD = 0.6 x 10(-6)). This affinity is in close agreement with previous results obtained using isolated brush-border membrane vesicles. Bovine lactoferrin inhibited the binding of porcine lactoferrin. Porcine transferrin, however, did not affect porcine lactoferrin binding significantly. Thus, lactoferrin binding is highly specific. When enterocytes were incubated with 125I-labeled lactoferrin at 37 degrees C, the amount of cell-associated radioactivity exceeded the surface binding capacity of the cells by almost fivefold. This finding agrees with the continuous binding and subsequent internalization of 125I-labeled lactoferrin. The isolated piglet enterocyte seems to provide a useful model for further studies of the mechanism of receptor-mediated absorption of lactoferrin.

Animals↗
PubMed1993

Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment.

Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.

Amino Acid Sequence↗
PubMed2001

Camel lactoferrin, a transferrin-cum-lactoferrin: crystal structure of camel apolactoferrin at 2.6 A resolution and structural basis of its dual role.

Camel lactoferrin is the first protein from the transferrin superfamily that has been found to display the characteristic functions of iron binding and release of lactoferrin as well as transferrin simultaneously. It was remarkable to observe a wide pH demarcation in the release of iron from two lobes. It loses 50 % iron at pH 6.5 and the remaining 50 % iron is released only at pH values between 4.0 and 2.0. Furthermore, proteolytically generated N and C-lobes of camel lactoferrin showed that the C-lobe lost iron at pH 6.5, while the N-lobe lost it only at pH less than 4.0. In order to establish the structural basis of this striking observation, the purified camel apolactoferrin was crystallized. The crystals belong to monoclinic space group C2 with unit cell dimensions a=175.8 A, b=80.9 A, c=56.4 A, beta=92.4 degrees and Z=4. The structure has been determined by the molecular replacement method and refined to an R-factor of 0.198 (R-free=0.268) using all the data in the resolution range of 20.0-2.6 A. The overall structure of camel apolactoferrin folds into two lobes which contain four distinct domains. Both lobes adopt open conformations indicating wide distances between the iron binding residues in the native iron-free form of lactoferrin. The dispositions of various residues of the iron binding pocket of the N-lobe of camel apolactoferrin are similar to those of the N-lobe in human apolactoferrin, while the corresponding residues in the C-lobe show a striking similarity with those in the C-lobes of duck and hen apo-ovotransferrins. These observations indicate that the N-lobe of camel apolactoferrin is structurally very similar to the N-lobe of human apolactoferrin and the structure of the C-lobe of camel apolactoferrin matches closely with those of the hen and duck apo-ovotransferrins. These observations suggest that the iron binding and releasing behaviour of the N-lobe of camel lactoferrin is similar to that of the N-lobe of human lactoferrin, whereas that of the C-lobe resembles those of the C-lobes of duck and hen apo-ovotransferrins. Hence, it correlates with the observation of the N-lobe of camel lactoferrin losing iron at a low pH (4.0-2.0) as in other lactoferrins. On the other hand, the C-lobe of camel lactoferrin loses iron at higher pH (7.0-6.0) like transferrins suggesting its functional similarity to that of transferrins. Thus, camel lactoferrin can be termed as half lactoferrin and half transferrin.

Animals↗
PubMed2006

Developmental gene expression of lactoferrin in duodenum and effect of weaning age on gene expression of lactoferrin in piglets.

Two experiments were conducted to evaluate duodenal gene expression of lactoferrin and effect of weaning age on mRNA expression of lactoferrin in piglets using semi-quantitative RT-PCR analysis. In experiment 1, a total of 15 female Duroc x Landrace x Yorkshire piglets of five groups, each group pigs at 1, 14, 28, 42 and 56 days of age were used to determine developmental gene expression of lactoferrin in duodenum. In experiment 2, a total of 18 female neonatal piglets were divided into three groups, which were weaned at 21, 28 and 35 days of age respectively. In each group, three piglets' duodena were sampled at 21, 28 and 35 days of age and the other three piglets' duodena were sampled 7 days after weaning in each group. The samples were collected for detecting the effect of weaning age on lactoferrin mRNA expression of piglets. The results show that lactoferrin mRNA levels decreased steadily in postnatal day 1-56. But only from day 28-42 (14 days after weaned), the levels of lactoferrin gene expression were decreased markedly (p < 0.05), and the difference of lactoferrin mRNA levels at other stages was not significant. This result suggested that weaning had an effect on gene expression of lactoferrin. The results of experiment 2 showed that when the piglets were weaned at 21-28 and 35 days of age respectively, the expression levels of lactoferrin were decreased by 77%, 53% and 59% at the seventh day after weaning. Our results showed that weaning significantly decreased lactoferrin mRNA expression of piglets.

Age Factors↗
PubMed1992

Interaction of lactoferrin and lipopolysaccharide (LPS): effects on the antioxidant property of lactoferrin and the ability of LPS to prime human neutrophils for enhanced superoxide formation.

Lactoferrin is a 77-kDa iron-binding protein to which a wide variety of divergent biologic functions have been ascribed. It has recently been reported that lactoferrin interacts with bacterial lipopolysaccharide (LPS) in such a fashion as to affect the binding of lactoferrin to myeloid cells. Two other potential interactions of LPS and lactoferrin were explored. Lactoferrin prevents hydroxyl radical formation by binding iron, even at low pH. Lactoferrin inhibited iron-catalyzed formation of hydroxyl radical in the presence of LPS at pH 7.4 and 4.5. Low concentrations of LPS can be used to "prime" neutrophils toward enhanced function, such as formation of stimulated superoxide anion. Lactoferrin inhibited LPS priming of neutrophils if LPS contamination of the protein (provided by commercial suppliers) was first reduced. Inhibition of LPS priming was observed whether apolactoferrin or iron-saturated lactoferrin was used. Similar inhibition of LPS priming was observed when neutrophils were incubated with other serum proteins (e.g., albumin, apotransferrin, or iron-saturated transferrin). These results show that LPS should not be expected to affect the free radical biology of lactoferrin, which is a crucial physiologic function of this protein. However, lactoferrin inhibits LPS priming, and this effect requires consideration in experimental models of inflammation.

Apoproteins↗
PubMed2002

Conditions influencing the in vitro antifungal activity of lactoferrin combined with antimycotics against clinical isolates of Candida. Impact on the development of buccal preparations of lactoferrin.

Lactoferrin, an iron-binding glycoprotein, is a potential agent for the treatment of oropharyngeal Candidiasis. The aim of the present study was to test the capability of lactoferrin, combined or not combined with conventional antifungal agents, to inhibit the growth of different Candida species under various experimental conditions to be of guidance in the development of a suitable pharmaceutical formulation containing lactoferrin. The anti-Candida activities of lactoferrin were considerably higher using RPMI instead of SLM as assay medium. They were moreover increased by raising the medium pH from 5.6 to 7.5. With the 'standard' antifungal agent fluconazole similar results were found as for lactoferrin, but the medium type and pH did not affect MIC values of amphotericin B. The addition of saliva to medium did not reduce the antifungal activities of the individual compounds. Synergistic inhibitory effects on Candida growth were found for combinations of lactoferrin and fluconazole or amphotericin B, irrespective of the medium type and pH, or the addition of saliva. This indicates that for treatment of oral Candidiasis a formulation containing lactoferrin seems appropriate; results may be optimized if the formulation is provided with buffer capacity to attain pH 7.5 in the mucosal fluid. The synergistic effects between lactoferrin and 'standard' antifungals indicate that combinations should be considered in such a formulation.

Antifungal Agents↗
PubMed2001

Characterization of glycans in a lactoferrin isoform, lactoferrin-a.

The presence of glycan at Asn-281 in bovine lactoferrin-a, which has a higher molecular weight than regular lactoferrin-b, was found in our previous study. The present work was performed to clarify the structures of the glycans linked to the five N-glycosylation sites in lactoferrin-a and to compare them with those of glycans linked to lactoferrin-b. In lactoferrin-a, the glycans linked to Asn-233 and Asn-545 were of the high-mannose type, whereas those present at Asn-368 and Asn-476 were complex-type ones. These glycans possessed heterogeneous structures. A comparative study of the glycans on bovine lactoferrin-a and bovine lactoferrin-b by HPLC showed that the structures of the glycans linked to Asn-368, Asn-476, and Asn-545 were very similar, the exception being the glycan linked to Asn-233. In addition, analysis of the structure of the glycan bound to Asn-281 present only in lactoferrin-a showed it possessed the heterogeneous structure of a complex-type glycan in which the structures Man3GlcNAc2, Man3GlcNAc4, Man3GlcNAc4Fuc are suggested to be present based on HPLC retention times only.

Animals↗
PubMed1994

The monocytic receptor for lactoferrin and its involvement in lactoferrin-mediated iron transport.

Several studies suggest biological functions of the iron-binding neutrophilic glycoprotein lactoferrin that imply an initial interaction with cells from the monocyte/macrophage family. Among these, an important role of lactoferrin as responsible for the inflammatory-induced blood hyposideremia and accumulation of iron in the monocyte/macrophage system has been suggested mainly based on experiments in rodents. In a series of experiments we have examined the binding of human lactoferrin to human monocytes. We have demonstrated the presence of a receptor binding including a high-affinity component and a low-affinity component. The affinity of the binding is compatible with a biological significance of this receptor (KD is about 10(-8) M, and the number of receptors about 10(6) per cell). More than 90% of the lactoferrin will dissociate from the cell. The binding is not truly reversible since lactoferrin will lose its receptor-binding property after dissociation from the cell. The only observed change in the molecule is a small decrease in isoelectric point from 8.9 to 8.8. Lactoferrin is able to translocate at least 50% of its bound iron to intracellular ferritin in monocytes. These findings are compatible with the idea that lactoferrin might be involved in the pathogenesis of the disturbances in iron metabolism observed during inflammation.

Biological Transport↗
PubMed2003

Potassium efflux induced by a new lactoferrin-derived peptide mimicking the effect of native human lactoferrin on the bacterial cytoplasmic membrane.

A 31-amino acid synthetic peptide (NH(2)-FFSASCVPGADKGQFPNLCRLCAGTGENKCA-COOH) was chemically synthesized based on the amino acid sequence of a region of human lactoferrin homologous to other sequences present in the N- and C-lobes of all members of the transferrin family proteins. The peptide, termed kaliocin-1, and lactoferrin showed a bactericidal effect in assays performed in low-ionic-strength conditions. This is the first time that it is shown that the antimicrobial effect of lactoferrin depends on the extracellular cation concentration. The antimicrobial effect of kaliocin-1 was lower than that of human lactoferrin, but their activities were inhibited by Na(+) or K(+) in a cation concentration-dependent manner. In addition, the peptide was able to mimic native lactoferrin, inducing K(+)-efflux and a selective dissipation of the transmembrane electrical potential of Escherichia coli cells without causing extensive damage to the outer and inner bacterial membranes. In contrast, the peptide, but not lactoferrin, was able to permeabilize different ions through liposomal membranes. The hypothetical interaction of kaliocin-1 with a bacterial membrane compound is discussed based in the different ion flux induced on cellular and artificial membranes as well as data from circular dichroism assays. Kaliocin-1 was not cytotoxic and could be a suitable model for the design of analogs able to mimic the antibacterial effect of human lactoferrin.

Amino Acid Sequence↗
PubMed2005

Lactoferrin expression by bovine ocular surface epithelia: a primary cell culture model to study lactoferrin gene promoter activity.

Tear lactoferrin, mainly secreted by the lachrymal glands, exerts a protective effect on the ocular surface, and an abnormal decrease of its production may lead to an increased risk of infection and pathological alterations of ocular surface epithelia. In this study we analyzed whether corneal and conjunctival epithelia could be an additional source of tear lactoferrin, and whether conjunctival epithelial cells in culture could be a suitable model system to address regulation of lactoferrin gene expression. Real-time PCR and Western immunoblotting showed that in bovines lactoferrin is indeed produced by these epithelia, and that the human lactoferrin promoter can direct the expression of a CAT reporter gene, thus indicating that these cells are a true source of lactoferrin, and may be used in vitro to study the regulation of lactoferrin expression.

Animals↗
PubMed1991

Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila.

We have been exploring the role of iron in the pathogenesis of the intracellular bacterial pathogen Legionella pneumophila. In previous studies, we have demonstrated that L. pneumophila intracellular multiplication in human monocytes is iron dependent and that IFN gamma-activated monocytes inhibit L. pneumophila intracellular multiplication by limiting the availability of iron. In this study, we have investigated the effect on L. pneumophila intracellular multiplication of lactoferrin, an iron-binding protein which is internalized via specific receptors on monocytes, and of nonphysiologic iron chelates which enter monocytes by a receptor-independent route. Apolactoferrin completely inhibited L. pneumophila multiplication in nonactivated monocytes, and enhanced the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication. In contrast, iron-saturated lactoferrin had no effect on the already rapid rate of L. pneumophila multiplication in nonactivated monocytes. Moreover, it reversed the capacity of activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron from the lactoferrin-lactoferrin receptor pathway. The capacity of iron-lactoferrin to reverse monocyte activation was dependent upon its percent iron saturation and not just its total iron content. Similarly, the nonphysiologic iron chelates ferric nitrilotriacetate and ferric ammonium citrate completely reverse and ferric pyrophosphate partially reversed the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron derived from nonphysiologic iron chelates internalized by monocytes independently of the transferrin and lactoferrin endocytic pathways. This study suggests that at sites of inflammation, lactoferrin may inhibit or promote L. pneumophila intracellular multiplication in mononuclear phagocytes depending upon its degree of iron saturation. In addition, this study suggests a potential role for PMN in host defense against L. pneumophila--providing apolactoferrin to infected monocytes--and it supports the concept that PMN and monocytes may cooperate in host defense against intracellular parasites and other pathogens.

Apoproteins↗
PubMed1998

Growth inhibitory effect of bovine lactoferrin to Toxoplasma gondii tachyzoites in murine macrophages: tyrosine phosphorylation in murine macrophages induced by bovine lactoferrin.

Previous studies have shown that lactoferrin induces growth inhibitory effects in mouse macrophages against intracellular Toxoplasma gondii, and these effects were not mediated by the oxygen-dependent and inorganic nitrogen-dependent pathway. To clarify the mechanism of anti-Toxoplasma gondii activity induced by lactoferrin, we examined whether lactoferrin promoted the phosphorylation of tyrosine residues in macrophage proteins. In immunoblotting assays using anti-[phosphorylated tyrosine] monoclonal antibody, phosphorylation of tyrosine residues was detected in protein(s) of approximately 30 kDa in macrophages incubated with lactoferrin. Inhibition of the lactoferrin-induced tyrosine-phosphorylation by genistein led to loss of the lactoferrin-induced growth inhibitory effect against the parasites. These findings suggest that lactoferrin induces tyrosine-phosphorylation in macrophages, and the tyrosine-phosphorylation seems to be associated with the induction of the growth inhibitory activity exerted against intracellular Toxoplasma gondii.

Animals↗
PubMedDate not supplied

[Immunochemical comparison of lactoferrin of human milk and lactoferrin of neutrophils].

Neutrophilic lactoferrin was isolated from the eluate of the SKN-K hemosorbent utilized for extracorporal blood detoxication. Specific antibodies were isolated from the antiserum obtained after immunization of rabbits using the neutrophilic lactoferrin preparation. Immunological identity of human milk lactoferrin, neutrophilic lactoferrin from blood serum of healthy persons and of the patients with esophagus cancer was exhibited by means of double radial immunodiffusion by Ouchterloni and by double cross immunoelectrophoresis using the antibodies against milk and neutrophilic lactoferrins. Thus, milk lactoferrin, antiserum and antibodies against the enzyme might be used as immunoreagents in studies of neutrophilic lactoferrin.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Lactoferrin-melanin interaction and its possible implications in melanin polymerization: crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-A resolution.

The concentration of melanin determines the intensity of colors of the skin and hair of animals. Melanin pigments are tyrosine-based polymers formed in melanocytes within specialized organelles called melanosomes. In order to understand the mechanism of melanin polymerization, lactoferrin, a basic protein with a pI value of 9.0, has been used to produce melanin. Lactoferrin is a monomeric iron-binding protein with a molecular weight of 80 kDa. The crystals of lactoferrin were soaked in a solution containing dihydroxyphenylalanine (DOPA) and tyrosinase enzyme. These crystals were used for X-ray intensity data collection. The intensity data were collected to 2.7-A resolution to an overall completeness of 91% with an R(sym) of 0.071. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions: a = 85.0 A, b = 99.8 A, c = 103.4 A. The structure was determined by molecular replacement method, using the model of diferric mare lactoferrin, and refined to an R-factor 0.215 (R(free) = 0.287) for all the data to 2.7-A resolution. The final model comprises 5,281 protein atoms from 689 amino acids, 2Fe(3+), 2CO(2-)(3) ions, 2 indole-5,6-quinone molecules (IQ), and 73 water molecules. Two IQ molecules, one in each lobe, bind to lactoferrin. In the C-lobe, the IQ binds in the iron-binding cleft, whereas in the N-lobe, it is located in the side pocket between two alpha-helices, filled with solvent molecules in the native iron-saturated mare lactoferrin. The IQ molecules interact with protein molecule mainly through glutamic acid in both lobes, without significant perturbation to the protein structure. The orientation of N- and C-lobes in the present structure is similar to that observed in the native iron-saturated protein. However, as a result of the binding of IQ molecules, the orientations of the domains N1, N2 and C1, C2 in the two cases differ slightly.

Animals↗