No abstract

SummaryLactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 Å cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its longrecognized antimicrobial properties.

Human lactoferrin (hLf) has considerable potential as a therapeutic agent. Overexpression of hLf in the fungus Aspergillus awamori has resulted in the availability of very large quantities of this protein. Here, the three-dimensional structure of the recombinant hLf has been determined by X-ray crystallography at a resolution of 2.2 A Ê . The ®nal model, comprising 5339 protein atoms (residues 1±691, 294 solvent molecules, two Fe 3+ and two CO 2À 3 ions), gives an R factor of 0.181 (free R = 0.274) after re®nement against 32 231 re¯ections in the resolution range 10±2.2 A Ê . Superposition of the recombinant hLf structure onto the native milk hLf structure shows a very high level of correspondence; the mainchain atoms for the entire polypeptide can be superimposed with an r.m.s. deviation of only 0.3 A Ê and there are no signi®cant differences in side-chain conformations or in the iron-binding sites. Dynamic properties, as measured by B-value distributions or iron-release kinetics, also agree closely. This shows that the structure of the protein is not affected by the mode of expression, the use of strainimprovement procedures or the changes in glycosylation due to the fungal system.

The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.

The contribution of the conserved His ligand to iron binding in transferrins has been addressed by site-directed mutagenesis and X-ray crystallographic analysis. His 253 in the N-terminal half-molecule of human lactoferrin, LfN (residues 1-333), has been changed to Gly, Ala, Pro, Thr, Leu, Phe, Met, Tyr, Glu, Gln, and Cys by oligonucleotide-directed mutagenesis. The proteins have been expressed in baby hamster kidney cells, at high levels, and purified. The results show that the His ligand is essential for the stability of the iron binding site. All of the substitutions destabilized iron binding irrespective of whether the replacements were potential iron ligands or not. Iron was lost below pH approximately 6 for the Cys, Glu, and Tyr mutants and below pH 7 or higher for the others, compared with pH 5.0 for LfN. The destabilization is attributed to both steric and electronic effects. The importance of electronic effects has been shown by the crystal structure of the H253M mutant, which has been determined at an effective resolution of 2.5 A and refined to a final R factor of 0.173. The iron atom is changed from six-coordinate to five-coordinate; the Met 253 side chain is not bound to iron even though there appears to be no steric barrier. This is attributed to the poorer affinity of the thioether ligand for Fe(III) compared with imidazole nitrogen. The decreased stability of the iron binding is attributed solely to the loss of the His ligand as the protein conformation and interdomain interactions are unchanged.