Investigation into the Acyl Chain Packing of Endotoxins and Phospholipids under Near Physiological Conditions by WAXS and FTIR Spectroscopy

Brandenburg, Klaus; Funari, Sérgio S.; Koch, Michel H. J.; Seydel, Ulrich

doi:10.1006/jsbi.1999.4186

Cited by 62 publications

(59 citation statements)

References 35 publications

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“…Aggregate Structure of pLA-Small angle diffraction patterns of pLA, in the absence and presence of ␣␣Hb, were recorded at 37°C and at 90% water content, to determine the supramolecular aggregate structure of lipid A (28,31,32). The diffraction pattern of pure pentaacyl lipid A was indicative of a multilamellar structure, which can be concluded from the occurrence of the periodicity at d l ϭ 4.91 nm and the fourth order (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

Brandenburg

Garidel

Andrä

et al. 2003

Journal of Biological Chemistry

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The interaction of purified ␣␣ cross-linked hemoglobin (␣␣Hb) with a pentaacylated mutant lipopolysaccharide (pLPS) and the corresponding lipid A (pLA) was studied biophysically and the effects correlated with data from biological assays, i.e. cytokine induction (tumor necrosis factor-␣) in human mononuclear cells and the Limulus amebocyte lysate assay. Fourier transform infrared spectroscopic and Zeta-Sizer experiments indicated an electrostatic as well as a non-electrostatic binding of ␣␣Hb to the hydrophilic and to the hydrophobic moieties of the endotoxins with an increase of the inclination angle of the pLA backbone, with respect to the membrane surface, from 25°to more than 50°. Small angle synchrotron radiation x-ray diffraction measurements indicated a reorientation of the lipid A aggregates from a multilamellar into a cubic structure as a result of ␣␣Hb interaction. Thus, in the absence of ␣␣Hb, the molecular shape of the pentaacyl samples was cylindrical with a moderate inclination of the diglucosamine backbone, whereas, in the presence of the protein, the shape was conical, and the inclination angle was high. The cytokine-inducing capability in human mononuclear cells, negligible for the pure pentaacylated compounds, increased markedly in the presence of ␣␣Hb in a concentration-dependent manner. In the Limulus assay, the pentaacylated samples were active a priori, and their activity was enhanced following binding to ␣␣Hb, at least at the highest protein concentrations. The data can be understood in the light of a reaggregation of the endotoxins because of ␣␣Hb binding, with the endotoxin backbones then readily accessible for serum and membrane proteins. By using fluorescence resonance energy transfer spectroscopy, an uptake of the endotoxin-Hb complex into phospholipid liposomes was observed, which provides a basis for cell activation.

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Section: Resultsmentioning

confidence: 99%

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

Brandenburg

Garidel

Andrä

et al. 2003

Journal of Biological Chemistry

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“…Details of the data acquisition and evaluation system can be found elsewhere (9). The diffraction patterns were evaluated as described previously (12) by assigning the spacing ratios of the main scattering maxima to defined three-dimensional structures. The lamellar and cubic structures are most relevant here.…”

Section: Methodsmentioning

confidence: 99%

Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin

Andrä¹,

Koch

Bartels

et al. 2004

Antimicrob Agents Chemother

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NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues. It adopts an amphipathic, ␣-helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids. We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays. As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains. NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one. This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system. Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive. This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances.

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“…The s axis was calibrated with Ag-behenate, which has a periodicity of 58.4 Å. The diffraction patterns were evaluated as described previously (6), assigning the spacing ratios of the main scattering maxima to defined three-dimensional structures. The lamellar and cubic structures are most relevant here and are characterized by the following features.…”

Section: Isothermal Titration Calorimetry (Itc)mentioning

confidence: 99%

New Antiseptic Peptides To Protect against Endotoxin-Mediated Shock

Gutsmann

Rázquin-Olazarán

Kowalski

et al. 2010

Antimicrob Agents Chemother

109

147

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Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.The life-threatening clinical consequences of sepsis and septic shock arise from recognition of microbial immunostimulatory molecules by the hosts' professional immune cells and the release of hemodynamically active mediators. The most potent immunostimulatory constituents are part of the microbial cell envelope, such as lipopolysaccharide (LPS) or lipoproteins. They are released continuously due to cell growth and division and massively liberated as a consequence of the attack of the immune system. In the case of Gram-negative bacteria, the most potent factor is LPS, which, therefore, is also called an endotoxin. LPS concentrations in blood serum as low as 1 ng/ml are able to cause sepsis. Septic shock resulting from bacterial infection remains a frequent cause of death, particularly in intensive care units, with more than 200,000 people dying each year in the United States alone. Death by septic shock can happen despite appropriate broad-range antibiotic treatment, which may kill bacteria but is not only incapable of neutralizing immunostimulatory LPS but also may promote its release into circulation (11).The response of mammalian cells to LPS is initiated by its interaction with serum proteins such as lipopolysaccharidebinding protein (LBP) and specific receptors and/or binding proteins of immune cells such as soluble CD14 (sCD14) and membrane-bound CD14 (mCD14), which finally leads to cell activation through the Toll-like receptor 4 (TLR4)-MD-2 pathway (31). The hydrophobic moiety of LPS, lipid A, anchoring LPS to the bacterial outer membrane, constitutes the "endotoxic principle" of LPS (24). Enterobacterial lipid A consists of a diglucosamine backbone phosphorylated at positions 1 and 4Ј, to which six acyl chains are linked at positions 2,3 a...

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Investigation into the Acyl Chain Packing of Endotoxins and Phospholipids under Near Physiological Conditions by WAXS and FTIR Spectroscopy

Cited by 62 publications

References 35 publications

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin

New Antiseptic Peptides To Protect against Endotoxin-Mediated Shock

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