Properties of the iron-binding site of the N-terminal lobe of human and bovine lactotransferrins. Importance of the glycan moiety and of the non-covalent interactions between the N- and C-terminal lobes in the stability of the iron-binding site.

Abstract

The recent determination by X-ray diffraction of the tridimensional structure of human lactotransferrin has underlined the presence of two lobes, each composed of two domains, I and II, as well as the involvement of five ligands in the binding of iron. Only one of the ligands (Asp-61) is located in domain I (residues 1-90 and 252-320), while the others [two tyrosine, one histidine and one (bi)carbonate ion linked to an arginine residue] belong to domain II (residues 91-251). On the basis of these data and of our previous results concerning the isolation of the 30 kDa N-tryptic fragment (residues 4-281) and the 20 kDa N2-glycopeptide (N-terminal domain II; residues 91-253) from human and bovine lactotransferrins, we have compared the iron-binding properties of these two fragments. The results demonstrate that Asp-61, which is missing from domain II, does not take part in the stability upon protonation of the iron complex of both human and bovine lactotransferrins. Furthermore, by comparing the iron-binding properties of human and bovine lactotransferrins to those of isolated 30 kDa N-tryptic and 50 kDa C-tryptic fragments and of the reassociated N,C-tryptic complex of both proteins, it has been shown that the non-covalent interactions which occurred between the two lobes of lactotransferrins and in the reassociated N,C-tryptic complex can explain in part the high affinity of lactotransferrins for iron. Finally, deglycosylation experiments on the 30 kDa N-tryptic fragment and N-terminal domain II from human and bovine lactotransferrins demonstrate that full removal of the glycan moiety leads to the loss of iron-binding capacity and so underlines the importance of the glycan moiety in the stability upon protonation of the N-terminal iron-binding site of both lactotransferrins.