| It is probable that the bilobate glycoprotein lactoferrin plays an important role in the antimicrobial activities at mucosal surfaces and in neutrophils. Its high affinity, coordinate iron binding properties likely contribute to the diverse functions proposed for this protein. Three discrete structural features have been ascribed with microbicidal activity including the iron and anion binding domains in the clefts formed by each lobe, and the N-lobe surface motif rich in basic amino acid residues (lactoferricin). The C-lobe of lactoferrin, generated by enzyme cleavage, was purified free of lactoferricin containing fragments based on heparin affinity. C-lobe, but not lactoferricin-containing fragments had bactericidal activity against S. mutans.;Molecular techniques were used to selectively mutate the discrete residues involved in coordinate binding. In an Escherichia coli expression system, the bacteria containing wild type recombinant human lactoferrin (wt-rhLF) plasmid was growth retarded and failed to express rhLF. In contrast, bacteria containing N-lobe half molecule plasmid grew normally and expressed the N-lobe as a GST fusion protein. These data suggested that intact LF, but not the N-lobe had antibacterial function against E. coli. Single amino acid substitutions in the C-lobe critical to anion binding (methionine for arginine-477) or iron binding (phenylalanine for tyrosine-540) permitted growth equivalent to that obtained with cells containing vector only. The baculovirus expression system was adapted to produce wt-rhLF fulfilling the properties of native LF. Single amino acid substitutions in the C-lobe residues involved in coordinate binding (R477M or Y540F, but not D407S) permitted enhanced protein expression, but did not discernibly compromise the ability of the proteins to bind iron. All of the expressed proteins from the baculovirus system were capable of killing in the apo, but not iron-saturated forms.;ApoLF (iron-free/carbonate-displaced) was shown to inhibit the expression of arginine-specific cysteine proteases of Porphyromonas gingivalis . The ineffectiveness of iron/carbonate-saturated LF and carbonate-saturated LF indicated that the iron and anion binding properties were important to this antibacterial function. The data from the three bacterial systems were consistent with the hypothesis that iron and anion binding ligands of LF were the functional domains involved in each of these antibacterial activities. |
