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 in fection 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 inte ractions, solid-phase ligand binding assays were performed with hLF fr om human milk (natural hLF) and N-terminally deleted hLF variants. Iro n-saturated and natural hLF bound equally well to heparin, lipid A, hL Z and DNA. Natural hLF lacking the first two N-terminal amino acids (G ly(1)-Arg(2)) showed reactivities of one-half, two-thirds, one-third a nd one-third towards heparin, lipid A, hLZ and DNA respectively compar ed with N-terminally intact hLF. A lack of the first three residues (G ly(1)-Arg(2)-Arg(3)) decreased binding to the same ligands to one-eigh th, one-quarter, one-twentieth and one-seventeenth respectively. No bi nding occurred with a mutant lacking the first five residues (Gly(1)-A rg(2)-Arg(3)-Arg(4)-Arg(5)). An anti-hLF monoclonal antibody (El1) tha t 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 an d DNA. The role of limited N-terminal proteolysis of hLF in its anti-i nfective and anti-inflammatory properties is discussed.