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乳鐵蛋白(萃取液)
DESCRIPTION:
Lactoferrin is a glycoprotein that belongs to the iron transporter or transferrin family. It was originally isolated from bovine milk, where it is found as a minor protein component of whey proteins. Lactoferrin contains 703 amino acids and has a molecular weight of 80 kilodaltons. In addition to its presence in milk, it is also found in exocrine secretions of mammals and is released from neutrophil granules during inflammation.
Lactoferrin is considered a multifunctional or multi-tasking protein. It appears to play several biological roles. Owing to its iron-binding properties, lactoferrin is thought to play a role in iron uptake by the intestinal mucosa of the suckling neonate. That is, it appears to be the source of iron for breast-fed infants. It also appears to have antibacterial, antiviral, antifungal, anti-inflammatory, antioxidant and immunomodulatory activities.
Three isoforms of lactoferrin have been isolated: lactoferrin-alpha, lactoferrin-beta and lactoferrin-gamma. Lactoferrin-beta and lactoferrin-gamma have RNase activity, whereas lactoferrin-alpha does not. Receptors for lactoferrin are found in monocytes, lymphocytes, neutrophils, intestinal tissue and on certain bacteria. Lactoferrin is abbreviated LF and Lf. Bovine lactoferrin is abbreviated bLF.
Bovine lactoferrin, derived from whey proteins, is marketed as a nutritional supplement. Supplemental lactoferrin typically contains low amounts of iron.
ABSTRACT:
Tryptase, a serine protease released exclusively from activated mast cells, has been implicated as a potential causative agent in asthma. Enzymatically active tryptase is comprised of four subunits, and heparin stabilizes the associated tetramer. Lactoferrin, a cationic protein released from activated neutrophils, binds tightly to heparin, therefore we investigated lactoferrin as an inhibitor of tryptase and found that it is both a potent (Ki' is 24 nM) and selective inhibitor. Size exclusion chromatography studies revealed that lactoferrin disrupted the quaternary structure of active tryptase. Lactoferrin was tested in an allergic sheep model of asthma; aerosolized lactoferrin ( 3 ml phosphate-buffered saline, 0.5 h before as well as 4 and 24 h after inhalation challenge by Ascaris suum) abolished both late-phase bronchoconstriction (no significant increase in specific lung resistance 4 to 8 h following provocation, p < 0.05 versus vehicle treatment) and airway hyperresponsiveness (no detectable increase in airway sensitivity to carbachol challenge 24 h after antigen challenge, p < 0.05 versus vehicle). These data suggest tryptase involvement in both late-phase bronchoconstriction and airway hyperreactivity and furthermore suggest that a physiological function of neutrophil lactoferrin is the inhibition of tryptase released from mast cells.
Figure 1. Effects of lactoferrin on allergen-induced bronchoconstriction in sheep. Four sheep were treated with of lactoferrin or vehicle (3 ml PBS), 0.5 h before and 4 and 24 h after antigen challenge. Early- and late-phase specific lung resistance and airway responsiveness to carbachol were measured as described in METHODS. Statistical significance of the data was determined using paired t-test analysis.
RESULTS:
To determine the effect of lactoferrin on tryptase, enzyme (0.5 nM) was incubated with varying concentrations of lactoferrin for 1 h and residual enzyme activity was determined. As shown in Figure2, lactoferrin is a potent inhibitor of tryptase activity at a heparin concentration of 25 ng/ml (a 1fold molar excess of heparin to tryptase assuming an average molecular weight of 5,000). The calculated Ki' of lactoferrin is 24 nM. The results are similar for human lung tryptase and tryptase from HMC-1 cells. There is no detectable tryptase inhibition by apo-transferrin (which has 85% sequence homology with lactoferrin but no heparin-binding domain) at a concentration of 1 mM (data not shown) and tryptase incubated without lactoferrin for 1 h is stable (< 10% loss of enzyme activity).
Figure 2. Inhibition of tryptase by lactoferrin. Lactoferrin (at the concentration indicated) was incubated with HMC-1 tryptase (0.5 nM) for 1 h in low phosphate buffer with heparin . Enzyme activity was measured as described in METHODS and used for the calculation of Ki'. The enzyme activity of tryptase pre-incubated for 1 h in the absence of lactoferrin represents 100% enzyme activity.
To determine the importance of a heparin-lactoferrin interaction to the mechanism of enzyme inhibition, tryptase was incubated for 1 h with lactoferrin in the presence of a 200,000-fold molar excess of heparin . The results (Figure 2) indicate that excess heparin protects tryptase activity from the effects of lactoferrin.
The kinetic mechanism of tryptase inhibition by lactoferrin was determined. A double reciprocal plot (Figure 3) of the inhibition data indicates that lactoferrin appears to be a non-competitive inhibitor (Km of substrate is unchanged in the presence of inhibitor) when pre-incubated with tryptase for 1 h prior to the addition of substrate.
Figure 3. Double-reciprocal plot of the effect of lactoferrin on tryptase activity. HMC-1 tryptase (0.5 nM) and lactoferrin (at the concentration indicated) were mixed and pre-incubated for 1 h in low phosphate buffer prior to the addition of substrate (0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.65, 0.7, and 0.8 mM). The values of Km, Ki, and Kis are calculated to be 1.0 mM, 65 nM, and 67 nM, respectively.
The capacity of lactoferrin to inhibit other trypsin-like serine proteases (in addition to tryptase) was also investigated (Table 1). Lactoferrin is a poor inhibitor of trypsin, thrombin, plasmin, and rat tryptase (35) enzymes that do not require heparin for activity.
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TABLE 1 |
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Enzyme* |
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Human Tryptase |
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24 |
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Rat Tryptase |
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546,000 |
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Trypsin |
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> 1,000,000 |
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Thrombin |
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> 1,000,000 |
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Plasmin |
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> 1,000,000 |
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* Enzyme (human tryptase, 0.5 nM; rat tryptase, 2.5 nM; trypsin, 15.7 nM; thrombin, 12.9 nM; plasmin, 7.6 nM) was incubated with varying concentrations of lactoferrin for 1 h in Tris assay buffer and the reaction was started by the addition of substrate (Tosyl-Gly-Pro-Lys-pNA, 0.5 mM final concentration). |
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Because tryptase inhibition by lactoferrin appeared to be mediated via a heparin-dependent mechanism, the effect of lactoferrin on the stability of the tryptase tetramer was assessed by use of size-exclusion chromatography. Size-exclusion is useful for separating and measuring relative amounts of tryptase tetramer (retention time, 7.5 min) and monomer (retention time, 9.5 min). A mixture of tryptase (340 nM) and porcine heparin (24 µg/ml) was incubated in the presence (15 µM) and absence of lactoferrin for 18 h. Samples were removed from both solutions at 1, 3, and 18 h after mixing for the determination of residual tryptase activity (Table 2) and tryptase tetramer decomposition (Figure 4).
Figure 4. Effect of lactoferrin on tryptase quaternary structure. Solutions of heparin (24 µg/ml) and tryptase (340 nM) with and without lactoferrin (15 µM) were incubated in low phosphate buffer at room temperature for 1, 3, and 18 h. Aliquots were removed and injected onto a size-exclusion column. Fluorescence ( ex, 280 nm; em, 335 nm) is shown on the ordinate and the retention time (in minutes) on the abscissa. The chromatograms displayed are samples of: (A) tryptase incubated for 1 h in the absence of lactoferrin, (B ) tryptase incubated for 1 h in the presence of lactoferrin, and (C ) tryptase incubated with lactoferrin for 18 h. (D) Western slot blot detection for tryptase protein using a portion (500 µl) of the fractions collected from experiments A, B, and C.
The chromatograph (Figure 4, panel A) of the control mixture (no lactoferrin) following a 1 h incubation, shows that tryptase is a mixture of tetramer (large peak) and monomer (small peak). It is not known if the tryptase monomer present in the preparation (25% of the tryptase protein, Table 2) reflects enzyme dissociation in solution or on the column or the presence of significant levels of contaminating inactive monomer in the tryptase preparation.
The chromatograph (Figure 4, panel B) of a sample from the mixture of tryptase and lactoferrin incubated for 1 h indicates that a substantial conversion of tryptase tetramer to monomer accompanies tryptase inhibition by lactoferrin (49% remaining tetramer, 33% inhibition, Table 2). In a sample from the same reaction mixture, following 18 h of incubation (Figure 4, panel C), the peak corresponding to tryptase tetramer is absent and the disappearance of tetramer is concomitant with a 90% loss of enzyme activity (Table 2).
Immunoblot assay (with a rat anti-human tryptase monoclonal antibody) of samples from fractions collected throughout the chromatographic separations (Figure 4, panel D) confirmed that both the 7.5 min peak and the 9.5 min peak are derived from tryptase; no other fractions contained tryptase protein. The fraction that corresponded to the tetramer had substantial proteolytic activity; whereas, very low but measurable enzyme activity was found in the fraction in which monomer eluted.
Lactoferrin was tested in the allergic sheep model of asthma (39). These sheep develop early- and late-phase bronchoconstriction and an associated increase in bronchial responsiveness following antigen (Ascaris suum) challenge. Sheep treated with lactoferrin (10 mg in 3 ml of phosphate buffered saline by aerosol), 0.5 h before and 4 h and 24 h following antigen challenge had substantial reductions in both late-phase bronchoconstriction (4 h to 8 h following antigen challenge, Figure 1) and airway hyperresponsiveness to carbachol (24 h following antigen challenge, Figure 1). The magnitude of the early phase (0 to 4 h) was not significantly reduced by lactoferrin treatment; however, the duration of the early phase bronchoconstriction appeared to be shortened as a result of treatment.
Three-dimensional structure analyses of lactoferrin from several species – human, bovine, buffalo and mare – in different forms such as differic, dicupric, disamarium, oxalate-substituted diferric, oxalate-substituted dicupric and apo-lactoferrin have revealed various ways in which protein structure adapts to different structural and functional states. The lactoferrin molecule folds into two lobes, each of which is further divided into two domains. The metal-saturated form of lactoferrin adopts a closed conformation in all the species whereas the metal-free form (apo) of human lactoferrin has N-lobe in open conformation and the C-lobe is in closed conformation while in mare lactoferrin, both N- and C-lobes adopt closed conformations. On further extension to transferrins, in duck apo-ovotransferrin, both lobes are found in open conformations. Comparison of the differic, dicupric and disamarium lactoferrins has shown that different metals can, through variations in metal positions, have different stereochemistries and anion coordinations without significant changes in protein structure. Substitutions of oxalate for carbonate as seen in the structure of diferric dioxalate mare lactoferrin and in a hybrid dicupric complex with oxalate in one site in human lactoferrin show that larger anions can be accommodated by small side chain movements in the binding site. Lactoferrin also binds two molecules of melanin monomer, indole-5,6-quinone specifically suggesting its role in the mechanism of melanin polymerization. The multidomain/multilobe nature of lactoferrin also allows rigid body movements. Comparison of human, mare, bovine and buffalo lactoferrins, rabbit serum transferrin and duck ovotransferrin shows that the relative orientations of the two lobes in each molecule vary substantially. These variations may contribute to differences in their binding properties.
LACTOFERRIN is a monomeric, iron-binding 80 kDa glycoprotein. It belongs to the family of transferrin proteins. It serves to control iron levels in body fluids by sequestering and solubilizing ferric iron. Its presence in leucocytes and many exocrine secretions (e.g. milk, saliva, tears, mucosal and genital secretions) together with its ability to bind to a wide variety of cells has further been associated with other postulated functions. These include roles in the immune and inflammatory responses, as an antibacterial agent6, as a growth factor7 and as an antifungal agent.
The molecular properties of lactoferrin are well established, through a variety of chemical and physical studies amino acid sequence determinations and high resolution X-ray crystallographic analyses of lactoferrin from various sources. Our aim has been to determine the molecular basis for the remarkable binding properties of lactoferrin, including (1) the extremely tight (K 1020) but reversible binding of two ferric ions per molecule, (2) the absolute requirement for a bound anion (normally CO32–) with each Fe3+, (3) the acceptance by lactoferrin of a wide variety of other metal ions and anions in place of Fe3+and CO32–, (4) structural
comparison between iron-saturated and iron-free forms of lactoferrins, (5) structural comparison of lactoferrins containing different metal ions, (6) structural comparison of lactoferrins with different anions, (7) comparison of lobe orientations in lactoferrins, (8) comparison of interdomain orientations, (9) the characteristic differences between different lactoferrins and transferrins and (10) the structural and functional similarities relating lactoferrins (and related transferrins) to a group of periplasmic binding proteins involved in active transport and chemotaxis.
In order to throw some light on the aspects mentioned above, we describe the structures of lactoferrins, the differences found in the structures of various forms of lactoferrins and the relevance of these to the mechanisms of binding and release. The structural comparisons with transferrins and periplasmic binding proteins have also been presented briefly.
Experimental:
Purification of lactoferrin
Lactoferrin is present in high concentrations in the colostrum of higher animals. Lactoferrin was purified from fresh colostrum using a locally modified procedure as described earlier37. Diluted colostrum/milk was defatted by skimming. Skimmed milk was diluted twice with 0.05 M tris-HCl, pH 8.0. CM-Sephadex C-50 was added to it (7 g/l) and stirred slowly by mechanical stirrer for an hour. The gel was allowed to settle and the milk was then decanted. The gel was washed with excess of 0.05 M tris-HCl, pH 8.0, packed in a column (25 ? 2.5 cm) and washed with the same buffer containing 0.1 M NaCl which facilitated removal of impurities. Lactoferrin was then eluted with the same buffer containing 0.25 M NaCl. The protein solution was dialysed against an excess of triple-distilled water. The protein was again passed through CM-Sephadex C-50 column (10 ? 2.5 cm) pre-equilibrated with 0.05 M tris-HCl, pH 8.0 and eluted with a linear gradient of 0.05 M–0.3 M NaCl in the same buffer. Protein was concentrated by Amicon ultrafiltration cell. The concentrated protein was passed through Sephadex G-100 column (100 ? 2 cm) using 0.05 M tris-HCl buffer, pH 8.0. The purity of lactoferrin was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)38 (Figure 1).
Figure 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of buffalo lactoferrin. Lane a: molecular weight markers (Pharmacia): phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and a -lactalbumin (14.4 kDa); lane b: purified lactoferrin (80 kDa).
Structure:
LTF belongs to the transferrin family proteins (TF, melanotransferrin, ovotransferin, etc.). Its molecular mass is 80,000 u (80 kDa).
(ferric state). proteolysis produces lactoferricin, kaliocin-1 small peptides with antimicrobial activity.
LTF receptors have been found on brush-border cells, PMN, monocytes, and activated lymphocytes.
Why use Lactoferrin?
INFLUENCE OF LACTOFERRIN ON THE IMMUNE SYSTEM:
Introduction
l Lactoferrin has been shown to stimulate the immune system by modulating the activity of immune-system components during the course of infection.(1) Several studies have demonstrated the effect of oral administration of lactoferrin on the immune system.(1,2,3) Although the mechanisms by lactoferrin influences the immune system are mostly unknown, a variety of effects at a cellular and molecular level have been documented.
l Influence of Lactoferrin on cells of the Immune System.
Lymphocytes:
Oral administration of lactoferrin in mice resulted in an increase of immunoglobulin A and G in the intestinal fluid as well as proliferation of lymphocyte-producing cells.
l In patients with chronic hepatitis C, oral administration of lactoferrin may increase the percentage of T helper cells (Th0 and Th1) in the peripheral blood.
l Lactoferrin was shown to promote the preferential maturation of CD4 CD8 T cells to the T helper CD4 linage.
l Lactoferrin can upregulate the CD4 surface marker in the human Jurkat lymphocyte cell line.
l Oral administration of lactoferrin was shown to increase the number of CD4+ T cells and CD8 + T cells in the peripheral blood, small intestine and spleen of mice.
Phagocytes:
l Receptors for lactoferrin have been found on monocytes and macrophages and research suggests that lactoferrin helps to control the activity of these cells. Lactoferrin assits the phagocytic action of these cells by promoting the production of free radicals within the phagosome. The proposed mechanism involves lactoferrin providing iron to an oxygen radical-generating system.
Influence of Lactoferrin on complement Activation:
l Bovine lactoferrin was shown to bind to Streptococcus agalactiae and activate the classical pathway of complement by substituting for antibodies. The action of complement increases the susceptibility of the bacteria to the action of phgocytes ( a process known as opsonization ).
l The iron-building properties of lactoferrin serve to provide an anti-oxidative effect of the immune system. By removing iron, lactoferrin may inhabit the production of free radicals and diminish oxidative damage to tissues. Lactoferrin may also protect neutrophilic cells from lipid peroxidative damage.
Lactoferrin as an antioxidant:
Finally, lactoferrin is an antioxidant that scavenges free iron helping to prevent uncontrolled iron based free radical reactions, thus protecting certain cells from peroxidation. Though lactoferrin is both an iron scavenger and donor (depending on the cellular environment), it has been found to scavenge or donate iron at the appropriate times when the body is in need of the reaction. At normal physiological PH, lactoferrin binds iron tightly thus diminishing oxidative stress to tissues (from free radical production of iron). Apolactoferrin, but not hololactoferrin, has been shown to prevent lipid peroxidation. One study that examined the role of whey proteins, multi-fermented whey proteins and lactoferrin in oxidative stress made the bold statement, “We can conclude that whey protein, lactoferrin and multi-fermented whey are good candidates as dietary inhibitors of oxidative stress and should be considered as potential medicinal foods in various pathologies as HIV infection and cancer.”
New research: antiviral, antibacterial, antifungal:
Previous studies have found lactoferrin to be a powerful inhibitor of a wide range of viruses. Recently, lactoferrin was tested against the deadly hantavirus and was directly compared to the anti-viral drug ribavirin.1 The study found that lactoferrin treated and pretreated cells greatly suppressed hantavirus. Perhaps even more intriguing, it was found that a powerful synergism existed when lactoferrin was combined with ribavirin. The researchers concluded, “These results indicate that lactoferrin has anti-hantaviral activity in vitro and inhibition of virus adsorption to cells, which play an important role in revealing the anti-hantaviral activity of lactorferrin. This paper reports for the first time the anti-hantaviral effect of lactoferrin.”
Lactoferrin is an iron binding protein secreted directly by the epithelial acinar cells of the lacrimal glands. Lactoferrin is one of the key molecules which modulates the inflammatory response, controls cell growth, protects against infections and allogeneic recognition reactions. By sequestration of iron, this protein exerts firm control of bacterial flora and inhibits the iron catalyzed production of hydroxyl radicals thereby protecting mucosal surfaces from oxidative damage. Lactoferrin has been shown to play a role in myelopoiesis, primary antibody response, lymphocyte proliferation, cytokine production, ADDC, NK cell activity and regulation of complement activation.
The primary lacrimal gland proteins, lactoferrin (bacteriostatic, anti-inflammatory, growth regulator), lysozyme (antibiotic), and tear specific prealbumin (toxic waste cleaner), have been shown to have the same levels in stimulated and nonstimulated tears.
The lacrimal glands also produce other tear proteins including s-IgA (primary protector of mucosal epithelial surface) and epidermal growth factor (EGF), that have presumed biologic activity on the ocular surface. Lactoferrin and other lacrimal secretory proteins are neurally mediated. In basal (non-stimulated) tears, lactoferrin, lysozyme, tear specific pre-albumin, IgA, and s-IgA make up 93% of total tear protein.
Antibacterial Activity:
Secreted lactoferrin exerts antimicrobial action by chelation of iron or destabilization of bacterial membranes.6 Lactoferrin is considered a bacteriostatic in tear film, and is necessary for the formation of the principle natural ocular antibiotic, lysozyme. Studies have shown that in general, both lactoferrin and lysozyme elevate and decrease together in ocular surface disease. The cationic tear protein lactoferrin and lysozyme exhibit co-operative anti-staphylococcal properties. Binding of lactoferrin to lipoteichoic acid (LTA) is important in its synergy with lysozyme and interferes with the autolysins present on the LTA. Studies have suggested that, on binding to the anionic LTA of S. epidermidis, the cationic protein lactoferrin decreases the negative charge, allowing greater accessibility of lysozyme to the underlying peptidoglycan. Thus, locally produced lactoferrin bathes the ocular surface and sequesters iron potential pathogens.
Protection from Oxidative Stress:
Lactoferrin plays a key role in ocular defense because the eye is linked to the common mucosal immune system. The main biological properties of lactoferrin can be ascribed to its very strong binding of iron cations. Metabolites generated form activated neutrophils and macrophages are important mediators of initial tissue injury. These metabolites include superoxide and hydrogen peroxide. The hydroxyl radical, a highly reactive species, is formed through an iron-catalyzed reaction involving superoxide and hydrogen peroxide. Sequestration of iron by lactoferrin can inhibit the iron-catalyzed production of hydroxyl radicals thereby protecting mucosal surfaces from oxidative damage.
The severity of ocular inflammation and tissue damage may be mediated by the iron-catalyzed generation of hydroxyl radicals. Generation of hydroxyl radicals may lead to extensive tissue damage by producing peroxidation of cell membrane lipids, by oxidative damage to proteins, and by generation of other free radicals. Depleting free iron can inhibit the pro-inflammatory effects of hydroxyl radicals. Normal lactoferrin levels, binding the free iron, support this inhibition.
Reoxygenation injury after extended hypoxia is responsible for increased cellular damage in corneal epithelial cells. Studies have shown that lactoferrin is taken into corneal epithelial cells as an iron transporter or antioxidant against excessive free iron. This means that lactoferrin taken into cells of lymphocyte lineage may also play a role in transcriptional activation.
Lactoferrin has been shown to protect the cornea tissue from reoxygenation injury after extensive hypoxia. Lactoferrin in tears may have a physiological role in protecting the corneal epithelium from solar UV irradiation.
Anti-inflammatory:
The antioxidant activity of lactoferrin is caused by its iron-chelating properties within the cytoplasm. Lactoferrin plays a significant role as an anti-inflammatory by blocking the C pathway and preventing the formation of C3 convertase. Activation of the C3 pathway can lead to the generation of the anaphylatoxins, C3a and C5a causing an increase in capillary permeability. Lactoferrin modulates the inflammatory process by preventing the release of cytokines from monocytes and by regulating the proliferation and differentiation of immune cells.
Active site structure