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PubMed1986

Monoclonal antibody (II2C) to human lactoferrin inactivates the myelopoietic suppressive effect of human lactoferrin in vitro.

A mouse monoclonal antibody was prepared against purified and fully iron-saturated human breast milk lactoferrin (LF). This antibody was of the IgG1 subclass, and recognized LF biosynthesized in low-density normal human bone marrow cells and LF stored in normal human polymorphonuclear neutrophils. This antibody did not recognize purified and iron-saturated human transferrin. The antibody inactivated the suppressive effects of purified and iron-saturated human milk LF and LF present in crude extracts of normal polymorphonuclear neutrophils against the release of granulocyte-macrophage colony-stimulating factors from mononuclear blood leukocytes in vitro.

Animals↗
PubMed1980

Lactoferrin deficiency as a consequence of a lack of specific granules in neutrophils from a patient with recurrent infections. Detection by immunoperoxidase staining for lactoferrin and cytochemical electron microscopy.

Neutrophils from a boy suffering from recurrent infections were found to be totally deficient in specific granules when studied by electron microscopy. In contrast, myeloperoxidase-containing azurophil granules were increased in number. This deficiency of specific granules could be detected at the light-microscopic level using an immunocytochemical technique to demonstrate the absence of lactoferrin. Neutrophils also exhibited abnormal nuclear segmentation, nuclear clefts, an abnormally weak cytochemical reaction for alkaline phosphatase, and an increased number of mitochondria and ribosomes. Some granulocytic precursors were abnormal, and many of these cells were phagocytosed by macrophages in the bone marrow. Despite these multiple abnormalities and the history of severe pyogenic infection, the in vitro bactericidal capacity of the neutrophils was within normal limits, and normal degranulation of azurophil granules occurred following phagocytosis. The precise mechanism by which the deficiency of specific granules in this patient led to an enhanced in vivo susceptibility to infection therefore remains obscure. However, attention is drawn to the fact that in three previously described cases of specific granule deficiency a history of recurrent infections was present.

Bacterial Infections↗
PubMed1998

Lactoferrin. Antiviral activity of lactoferrin.

A series of native and chemically derivatized lactoferrins (Lfs) purified from milk and colostrum were assayed in vitro for their anti-HIV and anti-HCMV-cytopathic effects in MT4 cells and fibroblasts respectively. All Lfs from bovine and human milk or colostrum were able to completely block HCMV replication as well as inhibited HIV-1 induced cytopathic effects. Through acylation of the amino function of the lysine residues in Lf, using anhydrides of succinic acid or cis-aconitic acid, negatively charged Lf derivatives were obtained that all showed a strong antiviral activity against the HIV-1 in vitro. Acylated-Lf exhibited a 4-fold stronger antiviral effect on HIV-1 than the parent compound but the activity on HCMV was abolished. Peptide scanning studies indicated that the native Lf as well as acylated Lf strongly bind to the V3 domain of the HIV envelope protein gp120, with Kd values in the same concentration range as the in vitro IC50. Therefore, shielding of this domain, resulting in inhibition of the virus-cell fusion and entry of the virus in MT4 cells is the likely mechanism underlying the anti-HIV activity. In contrast, addition of positive charges to Lf through amination of the proteins resulted in an increased anti-HCMV activity and a loss of anti-HIV activity, with anti-HCMV IC50 values in the low micromolar concentration range. The N-terminal portion of LF appeared essential to this anti-HCMV effect. The specific distribution of positively and negatively charged domains in the molecule appears to be important in both the anti-HIV and anti-HCMV effects.

Animals↗
PubMed1998

Identification and analysis of a CA(2+)-dependent lactoferrin receptor in rat liver. Lactoferrin binds to the asialoglycoprotein receptor in a galactose-independent manner.

We identified a 45 kDa Ca(2+)-dependent Lf binding protein on rat hepatocytes. Dithiobis(sulfosuccimidylproprionate) (DTSSP)-crosslinked 125I-Lf to a 45 kDa adduct in a Ca(2+)-dependent manner on intact cells. The 125I-labeled crosslinked complexes were absent when either surface-bound 125I-Lf was stripped prior to crosslinking or an excess of unlabeled Lf was included in the DTSSP reaction. Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, and a 45 kDa polypeptide (p45) was eluted by EGTA. Anti-p45 sera blocked vigorously 125I-Lf endocytosis to intact rat hepatocytes, confirming that p45 functions as the Ca(2+)-dependent Lf receptor on hepatocytes. Two tryptic fragments of p45 showed 100% identity with internal sequences (Leu121-->Lys126 and Phe198-->Lys220) of the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Antisera against p45 and RHL-1 crossreacted equally well with each protein, and asialoorosomucoid blocked the binding of 125I-Lf to hepatocytes. We did not detect the minor subunits (RHL-2/3) of the rat asialoglycoprotein receptor in p45 preparations from Triton X-100-extracts of hepatocytes, and 125I-Lf bound to immobilized RHL-1 but not to RHL-2/3. Exoglycosidases were used to remove terminally-exposed NeuNAc and alpha- and beta-Gal from bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal Gal. Unexpectedly, deglycosylated Lf exhibited no loss in its ability to compete with unmodified Lf for binding to isolated hepatocytes. Moreover, beta-lactose but not sucrose competed vigorously for 125I-Lf endocytosis by hepatocytes, indicating that Lf binds at or near the carbohydrate-recognition domain of RHL-1. We conclude that RHL-1 is the Ca(2+)-dependent Lf receptor on hepatocytes and that it binds Lf in a Gal-independent manner.

Amino Acid Sequence↗
PubMed1991

Uptake of lactoferrin by mononuclear phagocytes inhibits their ability to form hydroxyl radical and protects them from membrane autoperoxidation.

Human mononuclear phagocytes do not contain the iron-binding protein lactoferrin that we have previously demonstrated inhibits the potential for human neutrophils to generate hydroxyl radical in the presence of an exogenous iron catalyst of the Haber-Weiss reaction. Previous work by other investigators has suggested that mononuclear phagocytes (monocytes and monocyte-derived macrophages (MDM] have the capacity to bind exogenous lactoferrin via lactoferrin-specific membrane surface receptors. Accordingly, we examined the possibility that uptake of iron-free (apo) lactoferrin by human mononuclear phagocytes could play a role in limiting the potential for generation of hydroxyl radical during the monocyte/MDM respiratory burst. When monocytes or MDM were incubated in the presence of apo-lactoferrin, cell-associated lactoferrin increased in proportion to the concentration of lactoferrin provided. Similar results were obtained with iron-loaded (diferric) milk lactoferrin. Consistent with the in vivo importance of these findings, we found that lactoferrin was intimately associated with human alveolar macrophages obtained by bronchoalveolar lavage. The fucose polymer fucoidan inhibited lactoferrin uptake whereas exogenous transferrin or MDM exposure to IFN-gamma was without effect. Scatchard binding analysis confirmed the presence of a lactoferrin-specific receptor with a calculated kDa of 3.56 x 10(-6) M and 3.4 x 10(7) binding sites per cell. Subcellular fractionation studies indicated that twofold more of the lactoferrin which became cell-associated over the 1-h incubation time could be found in the cytoplasmic fraction compared to the plasma membrane-containing fraction, consistent with previous evidence by others for internalization of lactoferrin by mononuclear phagocytes. When lactoferrin-loaded monocytes/MDM were incubated in lactoferrin-free media, evidence for release of lactoferrin was obtained by SDS-PAGE and immunoblot analysis, suggesting the presence of a recyclable pool of cell-associated lactoferrin. To assess the impact of lactoferrin loading on monocyte/MDM hydroxyl radical formation, lactoferrin-loaded phagocytes were stimulated with PMA in the presence of catalytic iron. Hydroxyl radical generation by lactoferrin-loaded cells was decreased to about 50% of control cells. Similarly, monocytes that had been lactoferrin-loaded demonstrated a 28% decrease in autooxidation of their membrane when stimulated in the presence of catalytic iron. These data suggest that lactoferrin binding may play an important role in maintaining optimal mononuclear phagocyte function and protecting adjacent tissue from untoward phagocyte-associated hydroxyl radical generation.

Cell Membrane↗
PubMed1993

Removal of 14 N-terminal amino acids of lactoferrin enhances its affinity for parenchymal liver cells and potentiates the inhibition of beta- very low density lipoprotein binding.

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 arginine residues at positions 2-5 (APM-lactoferrin). After intravenous injection into rats, 125I-labeled APM-lactoferrin was cleared within 10 min by the liver parenchymal cells (74.7% of the dose). In contrast to native lactoferrin, APM-lactoferrin was rapidly internalized after liver association (> 80% of the liver-associated radioactivity was internalized within 10 min). 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 native lactoferrin (Kd 10 microM; 20 x 10(6) sites/cell). Preinjection of rats with 20 mg of APM-lactoferrin/kg of body weight reduced the liver association of beta-very low density lipoprotein (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 versus 8.0 mg/ml). Selective modification of the arginines of APM-lactoferrin with 1,2-cyclohexanedione reduced the liver association by approximately 60% and abolished the capacity of APM-lactoferrin to inhibit the binding of 125I-labeled beta-VLDL in vitro. In conclusion, our data indicate that the four-arginine cluster of lactoferrin at positions 2-5 is involved in its massive, low affinity association of lactoferrin with the liver, possibly to proteoglycans, but is not essential for the inhibition of lipoprotein remnant uptake. The Arg-Lys sequence at positions 25-31, which resembles the binding site of apolipoprotein E, may mediate the high affinity binding of lactoferrin and block the binding of beta-VLDL to the remnant receptor with high efficiency.

Amino Acid Sequence↗
PubMed1990

Lactoferrin binding molecules in human seminal plasma.

During ejaculation, the iron binding protein lactoferrin binds to sperm and forms a major component of sperm-coating antigens. Physicochemical properties of lactoferrin in seminal plasma (SP) and on sperm differ from those of purified lactoferrin. These differences have been attributed to the binding of unknown seminal macromolecules to lactoferrin. We have studied lactoferrin binding molecules in SP. The SP samples were coated onto microtiter plates and tested for binding of biotinylated lactoferrin. SP was found to specifically bind biotinylated lactoferrin. This binding was competitively inhibited by coincubation with unlabeled lactoferrin but was not affected by control incubations done with human IgG or transferrin. Lactoferrin binding molecules in SP were biochemically characterized by using SDS-PAGE and ligand blotting. Biotinylated lactoferrin bound to SP molecules of approximately 120, 60 and 30 kDa. No binding was observed with biotinylated transferrin. The presence of molecules that associate with lactoferrin in SP was further studied by using crossed immunoelectrophoresis. Lactoferrin in SP immunoprecipitated as two peaks, one of which corresponded to purified lactoferrin. These results suggest that some lactoferrin molecules in SP are free and that others are associated with lactoferrin binding molecules. Binding of lactoferrin to lactoferrin binding molecules appears to change its physicochemical properties and thus could influence its biologic activity and its affinity to sperm.

Carrier Proteins↗
PubMed2001

Lactoferrin-binding proteins of Tritrichomonas foetus.

Tritrichomonas foetus is a common, sexually transmitted, protozoan parasite of cattle. It has an essential requirement for iron, which it obtains from host lactoferrin. However, specific lactoferrin-binding protein receptors have not yet been identified in T. foetus. To differentiate specific and nonspecific binding of lactoferrin, lactoferrin affinity chromatography and Western blotting was used to identify metabolically or surface-labeled T. foetus lactoferrin-binding proteins. Bovine lactoferrin was shown to bind more efficiently than human lactoferrin, and each of these bound much better than bovine transferrin. This is relevant because T. foetus is both species-specific and only infects the mucosal surface of the reproductive tract, which has little transferrin. Whereas the majority of lactoferrin binding was specific, competitive inhibition studies showed that nonspecific, charge-related binding of lactoferrin to T. foetus may also be involved. In the presence of bovine cervical mucus, binding of lactoferrin to T. foetus was diminished, suggesting that mucus has an effect on lactoferrin binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface biotinylated proteins affinity-purified on lactoferrin-Sepharose showed biotinylated bands at Mr values of 22, 49, 55, 72, and 155 kDa. Because lactoferrin-binding proteins may be susceptible to digestion by T. foetus extracellular cysteine proteinases, it is suspected that the 155-kDa protein is the specific lactoferrin-binding protein and that the lower-Mr lactoferrin-binding molecules may be fragmentation products that contain the lactoferrin-binding site; however, other interpretations are clearly feasible. It is possible that there may be multiple proteins or multimers of the same protein. In summary, the data showed that binding of lactoferrin to T. foetus may be regulated by an interplay of specific receptor interactions as well as by hydrophobic and charge-related interactions.

Animals↗
PubMed2000

Separation of lactoferrin-a and -b from bovine colostrum.

Bovine lactoferrin was separated into lactoferrin-a and lactoferrin-b from bovine colostrum. Lactoferrin-a was eluted at 0.38 M NaCl and lactoferrin-b was eluted at 0.43 M NaCl by carboxymethyl cation-exchange chromatography at pH 7.7, 0.05 M phosphate buffer. The molecular weights were estimated at 84,000 for lactoferrin-a and 80,000 for lactoferrin-b. Lactoferrin-a contents were 258.0 mg/L and lactoferrin-b contents were 524.3 mg/L of colostrum for cow 19. From colostrum to normal milk, total lactoferrin was from 17.1 to 129.4 mg/L during the normal lactational period; however, lactoferrin did not separate clearly into lactoferrin-a and lactoferrin-b. The lactoferrin-a measured from six cows was 258.0, 114.0, 112.8, 64.0, 59.7, and 22.4 mg/ L and the lactoferrin-b 524.3, 331.8, 184.7, 170.7, 129.3, and 44.0 mg/L, respectively. The average was 105.2 mg (31.3%) for lactoferrin-a and 230.8 mg (68.7%) for lactoferrin-b.

Animals↗
PubMed1989

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.

Animals↗