Lipopolysaccharide (LPS) Neutralizing Peptides Reveal a Lipid A Binding Site of LPS Binding Protein

Abstract

Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms. Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity. The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo. These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site. Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting.

High Affinity Endotoxin-binding and Neutralizing Peptides Based on the Crystal Structure of Recombinant Limulus Anti-lipopolysaccharide Factor

“…Compared to the LPS-binding affinity of the holoprotein rLALF (17), this represents approximately a 10-fold loss in binding affinity of the peptides. Decreased binding affinity was reported in other cases when binding constants of peptides representing domains of proteins were compared with those of the holoprotein, in particular for the LPS-binding proteins BPI (23) and LBP (26). This is in accordance with our results and supports the identification of the LPS-binding domain being located between amino acid residues 36 and 45.…”

“…This includes synthetic peptides derived from sequences of polymyxin B (20), Tachypleus anti-LPS factor (TALF) (21), rLALF (22), bactericidal/permeability-increasing protein (BPI) (23), CAP-18 (24,25), and lipopolysaccharide-binding protein (LBP) (26).…”

Synthetic Endotoxin-Binding Peptides Block Endotoxin- Triggered TNF-α Production by Macrophages In Vitro and In Vivo and Prevent Endotoxin-Mediated Toxic Shock

“…Two other endotoxin-binding proteins from mammals, namely bactericidal/permeability increasing protein (BPI) and LPS binding protein (LBP), were proposed to have a similar endotoxin binding site (21). Although unrelated to LALF, endotoxin-binding domains were identified in both proteins using synthetic peptides (2,22,23). The endotoxin-binding domains of all three proteins are functionally competent within LBP in domain exchange mutant proteins (24).…”

“…This includes synthetic peptides derived from sequences of polymyxin B sulfate (PMB) (25), Tachypleus anti-LPS factor (26), recombinant LALF (rLALF) (27), BPI (23), CAP-18 (28,29), and LBP (22).…”

Apolipoprotein CI enhances the biological response to LPS via the CD14/TLR4 pathway by LPS-binding elements in both its N- and C-terminal helix

“…Where indicated, cells were preincubated with apoCI 1-57 (0.05 and 5 µg/ml), anti-CD14 antibody, or isotype control monoclonal antibody (10 µg/ml; Biometec) for 30 min at 37°C, washed, and subsequently stimulated with LPS. The medium was collected, and TNF ␣ was determined in the medium using the commercially available mouse TNF ␣ -specifi c OptEIA TM proteins, such as BPI, LPS-binding protein (LBP) ( 25 ), lactoferrin (Lf) ( 26 ), apoE ( 27 ), and MD2 ( 3 ) ( Table 1 ). Similar to the previously identifi ed LPS-binding motif of apoCI, these alternating cationic/hydrophobic motifs within apoCI are highly conserved during evolution ( 28 ) and may well be involved in the LPS-binding properties of apoCI.…”