Lactoferrin iron release

The two sites also differ in their pH stability towards iron release. Experiments on serum transferrin showed that one site loses iron at a pH near 6.0, and the other at a pH nearer 5.0 (203, 204), giving a distinctly biphasic pH-induced release profile (Fig. 28). The acid-stable A site was later shown to be the C-terminal site (202). It is this differential response to pH, together with kinetic effects (below), that enables N-terminal and C-terminal monoferric transferrins to be prepared (200). Although the N-terminal site is more labile, both kinetically and to acid, the reasons are not necessarily the same the acid stability may depend on the protonation of specific residues (Section V.B) and is likely to differ somewhat from one transferrin to another in response to sequence changes. The biphasic acid-induced release of iron seen for transferrin is not shared by lactoferrin. Although biphasic release from lactoferrin, in the presence at EDTA, has been reported (205), under most conditions both sites release iron essentially together at a pH(2.5-4.0) several units lower than that for transferrin (Fig. 28). 

Fig. 28. The pH dependence of iron release from human serum transferrin (Tf), human lactoferrin (Lf), and the recombinant N-terminal half-molecule of human lactoferrin (Lfm). Also shown is a plot (dashed line) for the release of cerium from Ce4+-substituted lactoferrin, demonstrating the increased difference between the two sites for metal ions other than Fe3+.

These observations come together with the evolutionary comparisons, which show that the N-lobe is highly conserved, through all species, whereas the C-lobe has become diversified. In serum transferrins, ovotransferrins, and lactoferrins it releases iron less readily than the N-lobe, because of its lesser flexibility, whereas in melanotransfer-rin and hornworm transferrin it no longer binds iron at all. Perhaps C-site binding has only remained where a receptor mechanism exists to extract iron from this site ... 

Fig. 30. Residues at the back of the iron site, near the hinge region, that may be implicated in the stimulation or modulation of iron release. The interactions present in human lactoferrin and rabbit transferrin are compared. Where the conformations are different, lactoferrin residues are shown with solid bonds, transferrin, with open bonds. Where the residues differ in identity or number, those for transferrin are in parentheses.

The recombinant whole molecules are both expressed in glycosylated form, although the glycosylation patterns differ from the proteins isolated from natural sources. The recombinant human transferrin binds to receptors both in its glycosylated form and as a nonglycosylated mutant, showing that the carbohydrate is not required for receptor binding (230). Recombinant human lactoferrin shows identical spectroscopic properties and shows an identical profile of pH-dependent iron release when compared with human milk lactoferrin (231). 

The half-molecules differ somewhat, although this is not unexpected, because the properties of the whole molecules are an amalgam of those of their two slightly different sites. Moreover, the lactoferrin halfmolecule shows that its iron release properties are changed as a result of the loss of interactions from the other lobe (Ref. 49 see also Section V.B.2 and Fig. 28). The visible max for the transferrin half-molecule... 

Transferrins6 max Iron binding Lactoferrins max Iron release (pH)... 

FhuA and FepA will prove to be the reference structures for a large group of bacterial outer-membrane transporters that take up bacterial Fe3+-siderophores, Fe3+ released from host transferrin and lactoferrin, haem, and haem released from haemoglobin and haemopexin. It is assumed that all iron sources are transported... 

Human lactoferrin (HL) is a component of innate immunity. Human lactoferrin is an iron-binding protein found in milk, granulocytes and exocrine secretions. It is released during inflammation, has bactericidal effects and reduces cytokine production by binding to the lipid A portion of endotoxin (Appelmelk et al., 1994). 

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