Lactoferrin Fragments Research Articles

Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells.

Human milk is in several ways anti-inflammatory. This study investigates whether or not human milk lactoferrin (LF) in comparison with bovine LF can affect the IL-6 release from human cells. Human, as well as bovine, LF and a bactericidal pepsin-derived fragment of bovine LF (lactoferricin B) were found to suppress the IL-6 response in a monocytic cell line (THP-1) when stimulated by lipopolysaccharide (LPS). The suppression of bovine LF was similar to or higher than that of human LF. Lactoferricin B was the strongest inhibitor of the LPS-induced IL-6 response. A time-dependence regarding the inhibitory capacity of LF was found. For human LF, the strongest inhibition was observed when added 15-30 min after the addition of LPS. Addition of LF before the LPS induced an approximately 45% reduction of the IL-6 response. The results suggest an anti-inflammatory activity of both human and bovine LF, and of the LF fragment lactoferricin B through their suppressive effects on the cytokine release.

The immunosuppressive mini-domain of human lactoferrin.

It has been found that the disulphide-bridged 231-245 pentadecapeptide loop of the lactoferrin (LF) N-lobe contains a region of immunosuppressive activity. The activity resides within a thymopentin-like sequence (Arg-Lys-Pro-Val-Asp) of the loop. Peptides related to the 575-589 loop of the LF C-lobe differ in their immunomodulatory activity from those related to the 231-245 loop. We ascribe this difference to the replacement of the Asp residue in the 231-245 loop by Thr in the 575-589 loop. Two other fragments of LF which were studied, 27-34 and 309-315, do not manifest any activity in the DTH test (cellular immune response), but, on testing in vivo, stimulate the humoral immune response. The 27-34 fragment is related to the bactericidal and immunostimulative region of LF identified by Bellamy et al. [1]. Our results show that the LF molecule contains, not only the known immunostimulating mini-domain, but also a region endowed with immunosuppressive activity.

Characterization of the Glycosaminoglycan-Binding Region of Lactoferrin

Lactoferrin is a prominent component of neutrophil secondary granules and its blood concentration is increased in certain inflammatory diseases. Although the biochemical characterization of lactoferrin as an iron-binding protein has been well described, its physiological role in inflammation remains undefined. We examined the ability of lactoferrin to regulate glycosaminoglycan-accelerated thrombin-serine protease inhibitor (serpin) reactions. Lactoferrin effectively reduced the rate of thrombin-serpin (antithrombin and heparin cofactor II) reactions by three physiological glycosaminoglycans including heparin, heparan sulfate, and dermatan sulfate. An enzyme kinetics analysis showed that lactoferrin did not alter the apparent heparin-thrombin or the heparin-antithrombin dissociation constant values for the heparin-catalyzed thrombin-antithrombin reaction. However, the maximum reaction velocity at saturation with respect to either protein was markedly decreased by lactoferrin. The glycosaminoglycan-binding region of lactoferrin was analyzed following limited proteolysis using Staphylococcus aureus V 8 protease. Two lactoferrin fragments with M r′s of ∼8 and ∼11 kDa were purified based on their affinity to heparin-Sepharose. Amino acid sequence analysis demonstrated that both peptides were from the N-terminus. Although slightly less capable compared to intact lactoferrin, the lactoferrin peptides effectively neutralized heparin, heparan sulfate, and dermatan sulfate-catalyzed serpin-thrombin inhibition reactions. In addition, lactoferrin N-terminal peptides have approximately the same binding affinity to heparin-Sepharose as that of intact lactoferrin. Inspection of both the N-terminal amino acid sequence and the crystal structure of lactoferrin further supports the conclusion that lactoferrin is a novel glycosaminoglycan binding protein and that the putative glycosaminoglycan-binding site is localized to the N-terminus.

Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.

Lactoferrin is an iron-binding protein which is synthesized by mucosal epithelium and neutrophils and released by these cells in response to inflammatory stimuli. It promotes neutrophil aggregation and manifests iron-dependent and -independent antimicrobial properties in vitro. Since lactoferrin binds to glycosaminoglycans (GAGs) and sulfated polysaccharides can inhibit its clearance in vivo and in vitro, we sought to examine its interaction with the GAGs chondroitin sulfate and heparin. Amino-terminal sequencing of proteolytic fragments of human lactoferrin that were fractionated by GAG chromatography suggested that the amino-terminal 6 kDa of the secreted protein mediates its interaction with GAGs. Synthetic peptides were used to show that the first 33 residues of human lactoferrin can bind well to solid-phase or solution-phase GAGs. The first 33 residues bound fluoresceinamine-labeled heparin with an IC50 (611 nM) which approximated that of the intact protein (124 nM). In contrast, when the first six residues (GRRRRS) were removed from this peptide, it then bound poorly to heparin (IC50 = 49 microM). Our results suggest that the GRRRRS sequence at the amino terminus of human lactoferrin acts synergistically with an RKVR sequence at positions 28-31 to form the predominate functional GAG-binding site of human lactoferrin. Molecular modeling of the crystalline structure of lactoferrin supports a synergistic activity between these two sites since it shows that they juxtapose each other on the surface of the folded protein. Solid docking calculations indicate that they can form a cationic cradle as a binding site for chondroitin sulfate.

Potent Bactericidal Activity of Bovine Lactoferrin Hydrolysate Produced by Heat Treatment at Acidic pH

A hydrolysate of bovine lactoferrin produced by heat treatment under acidic conditions had antibacterial activity at concentrations of 10μg/ml in the culture medium. The optimal degree of hydrolysis for this activity was about 10%. Heat-treated lactoferrin, treated at pH 2.0 and 120°C for 15min and degree of hydrolysis of about 10%, had no Fe-binding capacity (0%) and less antigenicity (about 10–6) than untreated lactoferrin. Heat-treated lactoferrin increased in antibacterial activity, and the activity was maintained in an Fe-rich medium. After fractionation of heat-treated lactoferrin by reverse-phase HPLC, several peptide fractions were found that had strong antibacterial activity. It was suggested that lactoferrin latently contains at least one bactericidal domain that is activated upon release by limited acid hydrolysis of the protein. The bactericidal activity of the peptide fragments of lactoferrin was shown to have no relation to Fe chelation, in contrast with the antibacterial mechanism of native lactoferrin.

Origin of Intact Lactoferrin and Its DNA-Binding Fragments Found in the Urine of Human Milk-Fed Preterm Infants. Evaluation by Stable Isotopic Enrichment

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13C]leucine and [15N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [13C] leucine and [15N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed.

Heat Stability of Bovine Lactoferrin at Acidic pH

In order to develop a practical method for pasteurization of lactoferrin, the influence of pH on the heat stability of bovine lactoferrin was studied. Solutions (5%; wt/vol) of iron-free lactoferrin (apo-lactoferrin) in distilled water were adjusted to various pH values from 2.0 to 11.O and heated at various temperatures (80 to 120°C) for 5min. The heated samples were analyzed by HPLC and assayed for iron-binding capacity, antigenic activity, and bacteriostatic effect. The heated solutions of apo-lactoferrin gelled at neutral and alkaline pH. At acidic pH, lactoferrin in the heated samples remained soluble, and solutions were clear. The HPLC pattern of apo-lactoferrin heat-treated at pH 4.0 and 100°C was similar to that of unheated apo-lactoferrin. It resisted heating at pH 4.0 and 90°C for 5min without any significant losses of iron-binding capacity, antigenic activity, or antibacterial activity. These results indicate that apo-lactoferrin is very stable at pH 4.0 and high temperatures. It was considered that heating conditions of pH 4.0 and temperatures of 90 to 100°C for 5min were suitable as a practical method for pasteurization of lactoferrin. Moreover, the possibility of using an UHT method for more effective and efficient pasteurization of lactoferrin was demonstrated. Apo-lactoferrin treated at pH 2.0 or 3.0 and 100 or 120°C for 5min was apparently degraded, but the antibacterial activity was equal to or stronger than that of unheated apo-lactoferrin. This result suggests that some of the lactoferrin fragments produced by heat under acidic conditions have antibacterial activity.

Molecular Forms of Lactoferrin in Stool and Urine from Infants Fed Human Milk

The molecular forms of lactoferrin (LF) were examined in stools and urine collected at 2.5 or 5 wk of age from very low birth wt infants fed either a cow's milk formula or a fortified human milk preparation. LF was not found by Western blotting in excreta from infants fed cow's milk. In contrast, intact and fragmented forms of LF were detected in stools and concentrated urine of each infant who received human milk. Only intact LF was detected in the fortified human milk preparation, whereas many types of LF fragments were present in the stools and urine. The approximate molecular wt of the most prominent fragments were 44, 38, 34, and 32 kD. However, the stools also displayed lower molecular wt fragments that were not found in urines of those infants. The LF fragments in those excreta were similar in size to those produced in vitro by limited digestion of apo-LF with trypsin. Furthermore, fragments produced by in vitro proteolysis were immunoreactive in an ELISA for LF. Thus, the fragments of LF in stools of very low birth wt infants fed human milk appeared to be produced by in vivo proteolysis, and the close resemblance between the LF fragments in the stools and urine suggests that the urinary LF fragments originated in the gastrointestinal tract. It remains unclear, however, whether the whole LF molecules that were fragmented were derived solely from ingested LF in human milk or in part from LF produced by the infant in response to human milk feedings.

ウシラクトフェリンのトリプシン分解物と固定化ブルー色素との親和性

ウシラクトフェリンのトリプシン分解物と固定化ブルー色素との親和性

Fe-saturation and proteolysis of human lactoferrin: effect on brush-border receptor-mediated uptake of Fe and Mn

We have previously characterized a brush-border membrane receptor that facilitates iron uptake from human lactoferrin. The receptor is specific for human and monkey lactoferrin and does not recognize human transferrin or bovine lactoferrin. In this study, iron uptake from lactoferrin fragments was studied, as well as from lactoferrin partially saturated with iron. Brush-border membrane vesicles (BBMV) prepared from infant rhesus monkey small intestine efficiently accumulated iron from lactoferrin half-molecules, although competition experiments showed that intact lactoferrin has a higher affinity toward the receptor. Lactoferrin partially saturated with iron also effectively delivered iron to the receptor, whereas the affinity was lower than for lactoferrin saturated with iron. Lactoferrin also carries a large proportion of human milk manganese, and receptor-mediated uptake of lactoferrin-bound manganese into BBMV was demonstrated, although this complex had lower affinity than that found for iron-lactoferrin. Thus, although the receptor has a preference for intact iron-saturated lactoferrin, partially digested lactoferrin and partially iron-saturated lactoferrin can also deliver iron to the receptor. Therefore, these molecular species, which are likely to occur in the gastrointestinal tract of the infant, may contribute to the high degree of iron absorption from human milk lactoferrin.

Lactoferrin and transferrin fragments react with nitrite to form an inhibitor of Bacillus cereus spore outgrowth.

Tryptone is a pancreatic digest of casein which contains a heterogeneous mixture of substances that react with nitrite when heated in the presence of sodium thioglycolate to form a bacteriostatic agent which inhibits outgrowth of Bacillus cereus T spores. The substances which are precursors to the bacteriostatic agent can be fractionated on the basis of molecular size and charge and have properties which indicate that they are fragments of lactoferrin, an iron-binding glycoprotein. The bacteriostatic agent could also be formed directly from purified lactoferrin after it had been subjected to proteolysis. Transferrin, an analogous iron-binding protein found in animal serum, also showed these same properties. This system may be a useful model for studies of the mechanism and site of nitrite bacteriostatic action.