Identification of single amino acid residues essential for the binding of lipopolysaccharide (LPS) to LPS binding protein (LBP) residues 86–99 by using an Ala‐scanning library

Abstract

Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.

N-Linked Glycosylation at Asn3and the Positively Charged Residues within the Amino-Terminal Domain of the C1 Inhibitor Are Required for Interaction of the C1 Inhibitor withSalmonella entericaSerovar Typhimurium Lipopolysaccharide and Lipid A

“…This hypothesis also is consistent with the previous observations that the binding of LPS to most, if not all, other LPS-binding proteins is dependent on positively charged sites (2,3,15,21,25,27,33,34,36,42,47,54,57). Removal of the carbohydrate at Asn 3 very likely results in a conformational rearrangement that obscures the positively charged binding site.…”

“…The LPS binding domains of these and other proteins share sequence homology with LBP (3,10,12,19,25). Based on both synthetic peptide and site-directed mutagenesis experiments, Arg 94 and Lys 95 of LBP are required for LPS binding activity (25,42,47,54). A similar positively charged region of MD-2, a component of the macrophage LPS receptor, is responsible for binding to LPS (20,31,40,60).…”

“…Analysis of the role of the positively charged residues might be approached through the use of synthetic peptides. However, studies with other LPS-binding proteins have shown that the findings of experiments with substituted peptides frequently do not correspond to the results of experiments using mutated intact proteins (25,42,47). Furthermore, the only practical approach to analysis of the three individual N-linked sites was through the use of recombinant mutated proteins.…”

N-Linked Glycosylation Is Required for C1 Inhibitor-Mediated Protection from Endotoxin Shock in Mice

“…The smaller dLPA molecule may be able to more readily gain access to the binding site, whereas the larger LPS molecule would be unable to interact with the site. Binding of LPS (or dLPA) to many LPS-binding proteins is via interaction of the phosphate groups on the LPA with specific positively charged residues within the LPS-binding protein (1,3,14,17,20,21,25,26,28,31,35,39,41). C1INH has one Arg (at position 18) and 3 Lys residues (at positions 22, 30, and 55) within the amino-terminal domain.…”

Comparison of the effect of recombinant bovine wild and mutant lipopolysaccharide-binding protein in lipopolysaccharide-challenged bovine mammary epithelial cells

“…Genetic variation in the promoter region of the LBP gene is associated with the blood level of LBP and with the risk of developing gram-negative bacteremia and gram-negative bacteremia-related death after hematopoietic cell transplantation (HCT) . In addition, several alaninesubstituted synthetic LBP-derived peptides inhibited LPS-LBP interaction (Reyes et al 2002). A mutation affecting amino acid 98, which affects an exposed loop of LBP, has been described to confer a risk for several infectious and inflammatory diseases (Shukla et al 2011).…”