N-terminal stretch Arg(2), Arg(3), Arg(4) and Arg(5) of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA

Human lactoferrin (hLF), a protein involved in host defence against infection and excessive inflammation, interacts with heparin, the lipid A moiety of bacterial lipopolysaccharide, human lysozyme (hLZ) and DNA. To determine which region of the molecule is important in these interactions, solid-phase ligand binding assays were performed with hLF from human milk (natural hLF) and N-terminally deleted hLF variants. Iron-saturated and natural hLF bound equally well to heparin, lipid A, hLZ and DNA. Natural hLF lacking the first two N-terminal amino acids (Gly(1)-Arg(2)) showed reactivities of one-half, two-thirds, one-third and one-third towards heparin, lipid A, hLZ and DNA respectively compared with N-terminally intact hLF. A lack of the first three residues (Gly(1)-Arg(2)-Arg(3)) decreased binding to the same ligands to one-eighth, one-quarter, one-twentieth and one-seventeenth respectively. No binding occurred with a mutant lacking the first five residues (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)). An anti-hLF monoclonal antibody (El1) that reacts to an N-lobe epitope including Arg(5) completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg(2)-Arg(3)-Arg(4)-Arg(5), has a decisive role in the interaction of hLF with heparin, lipid A, hLZ and DNA. The role of limited N-terminal proteolysis of hLF in its anti-infective and anti-inflammatory properties is discussed.