Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution.

Abstract

Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram‐negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti‐LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. Here we present the high resolution structure of a recombinant LALF. It has a single domain consisting of three alpha‐helices packed against a four‐stranded beta‐sheet. The wedge‐shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and we propose that two mammalian LPS‐binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

“…The two typical structures indicated that EsALF was an amphipathic factor and its crystal structure possibly was a hairpin loop just like Limulus ALF [37]. Phylogenetic analysis further revealed that the EsALF was close matched to ALFs from crabs and shrimps (the bootstrap was 67% to shrimps and 40% to crabs).…”

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

“…Limulus anti-LPS factor (LALF) is a protein from the horseshoe crab with high potential to block endotoxin-mediated activities in animals (17)(18)(19)(20). Both BPI and LALF crystal structures are known, and the LALF analysis revealed a detailed view of a potential endotoxin-binding site (21). The site consists of the aa 31-52 and is characterized by an alternating series of positively charged and hydrophobic residues forming a positively charged amphipathic loop (21).…”

“…Both BPI and LALF crystal structures are known, and the LALF analysis revealed a detailed view of a potential endotoxin-binding site (21). The site consists of the aa 31-52 and is characterized by an alternating series of positively charged and hydrophobic residues forming a positively charged amphipathic loop (21). 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).…”

“…The site consists of the aa 31-52 and is characterized by an alternating series of positively charged and hydrophobic residues forming a positively charged amphipathic loop (21). 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).…”

“…31 and Table I). Analogues peptides derived from BPI (B-14) and LBP (H-14) were prepared based on the alignment as proposed by Hoess et al (21). Both, H-14 and B-14 were derived from amino acid regions 90 -101 of LBP or BPI, respectively, and comprise 12 aa from the endotoxin-binding domain as well as 2 cysteines at both ends to form a cyclic peptide after oxidation (Table I).…”