Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide

• Published in: Biochemical journal 1995-12, Vol.312 ( Pt 3) (3), p.839-845
• Main Authors: Elass-Rochard, E, Roseanu, A, Legrand, D, Trif, M, Salmon, V, Motas, C, Montreuil, J, Spik, G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Binding, Competitive; Cattle; Escherichia coli - chemistry; Humans; Lactoferrin - chemistry; Lactoferrin - metabolism; Lipopolysaccharides - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Structure, Secondary; Recombinant Proteins - metabolism; Trypsin - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3120839

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.