N-terminal stretch Arg2, Arg3, Arg4 and Arg5 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 (Gly1-Arg2) 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 (Gly1-Arg2-Arg3) 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 (Gly1-Arg2-Arg3-Arg4-Arg5). An anti-hLF monoclonal antibody (E11) that reacts to an N-lobe epitope including Arg5 completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg2-Arg3-Arg4-Arg5, 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.
van Berkel
P H
PH
Leiden Institute of Chemistry, Medical Biotechnology Department, Gorlaeus Laboratories, Leiden University, The Netherlands.
Geerts
M E
ME
van Veen
H A
HA
Mericskay
M
M
de Boer
H A
HA
Nuijens
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive / Q000276:immunology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007781:Lactoferrin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008050:Lipid A / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009113:Muramidase / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D008050:Lipid A; D008070:Lipopolysaccharides; D006493:Heparin; D004247:DNA; D001120:Arginine; D009113:Muramidase; D007781:Lactoferrin