Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.
The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.
Brandenburg
K
K
Forschungszentrum Borstel, Abteilung Biophysik, Germany.
Jürgens
G
G
Müller
M
M
Fukuoka
S
S
Koch
M H
MH
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry* / Q000378:metabolism*; D008033:Limulus Test; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011112:Polymyxin B / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014961:X-Ray Diffraction
D008050:Lipid A; D008070:Lipopolysaccharides; D008081:Liposomes; D010743:Phospholipids; D014409:Tumor Necrosis Factor-alpha; D007781:Lactoferrin; D011112:Polymyxin B