title: 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-alpha 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 micro m, 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.
authors.affiliation: Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Andrä
authors.forename: Patrick
authors.initials: P
authors.lastname: Garidel
authors.forename: Andreja
authors.initials: A
authors.lastname: Majerle
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
authors.forename: Richard
authors.initials: R
authors.lastname: Ridge
authors.forename: Erik
authors.initials: E
authors.lastname: Paus
authors.forename: Tom
authors.initials: T
authors.lastname: Novitsky
authors.forename: Michel H J
authors.initials: MH
authors.lastname: Koch
authors.forename: Klaus
authors.initials: K
authors.lastname: Brandenburg
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D001667:Binding, Competitive; D002152:Calorimetry, Differential Scanning; D004563:Electrochemistry; D031541:Fluorescence Resonance Energy Transfer; D006434:Hemocytes / Q000166:cytology / Q000378:metabolism; D006737:Horseshoe Crabs; D006801:Humans; D007447:Invertebrate Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008033:Limulus Test; D008050:Lipid A / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism*; D044367:Phase Transition; D010843:Pichia / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D007447:Invertebrate Hormones; D008050:Lipid A; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C034705:antilipopolysaccharide factor (Limulus); D011112:Polymyxin B