Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide

Abstract

Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-a induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a K d of 19 lM, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a K d in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS-ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form.Keywords: endotoxins; IR spectroscopy; LALF; LPS; tumor necrosis factor. Lipopolysaccharides (LPS) represent the main amphiphilic component of the outer leaflet of the outer membrane of Gram-negative bacteria. They are often referred to as endotoxins due to their ability to induce a variety of biological effects in mammals, in particular the production of proinflammatory cytokines [1]. At low endotoxin concentrations, the biological effects may be beneficial, as some cytokines such as tumour necrosis factor-a (TNFa) have been shown to possess antitumour activity. At higher endotoxin concentrations, however, the release of cytokines leads to an inflammatory response, eventually resulting in the septic shock syndrome [2].LPS consists of a sugar moiety comprising the O-specific chain, outer core and inner core covalently linked to the hydrophobic moiety of LPS, lipid A, which anchors the LPS molecule to the membrane [2]. The sugar moiety type and length depends on the strain of bacteria. Smooth strains have varying length O-specific chains, while rough mutants have no O-specific chain (Ra) and can also lack parts of the core regions (Rb-Re). Deep rough mutant LPS (LPS Re) comprises the inner core, which co...

Design, structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

“…These values are consistent with those of other active antimicrobial peptides obtained using fluorescence spectroscopy [25]. ITC experiments, carried out at 37°C, revealed that binding of the peptide to LPS is an exothermic process, driven by the electrostatic interactions between the positively charged peptide and the negatively charged LPS as observed for other antimicrobial peptides reported in the literature [51,53,54].…”

LptE binds to and alters the physical state of LPS to catalyze its assembly at the cell surface

“…We think it is noteworthy that many of these proteins, in particular LALF, bactericidal permeability increasing protein (BPI) (41), and LPS-binding protein (LBP) (42), possess functions that extend beyond the mere binding of LPS. It has been hypothesized that the eukaryotic structural homolog LALF participates in not only the binding of LPS but also in its transport to and insertion into phospholipids membranes as part of the Limulus host immune response (23,35). LBP and BPI are elongated in shape and contain a duplicated fold similar to that of LptE.…”

“…Therefore, LptE binds to and solubilizes LPS adhered to a CM3 chip. Because the structure of LptE shows homology to the eukaryotic protein LALF, which binds and transports LPS (23,35), we wondered whether LALF would show similar binding interactions with LPS in our SPR assay. In fact, LALF is also able to extract LPS from the chip surface (Fig.…”

Characterization of two isoforms of antiliopolysacchride factors (Sp-ALFs) from the mud crab Scylla paramamosain

“…Several studies have investigated the LPS binding ability of the recombinant ALF [23]. Besides, it was proposed that the bactericidal effect of ALF like ALFPm3 from shrimp was due to its binding activity to components of the bacterial cell-wall [24].…”