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

Abstract

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.

Biophysical Characterization of the Interaction of Endotoxins with Hemoglobins

“…Thus, we found, that cross-linked Hb is able to convert an inactive pentaacylated having a lamellar conformation lipid A into an endotoxically active cubic conformation [11]. There is evidence that the enhancement of the biological activity is also associated with a Hb-induced disaggregation of LPS [11,12].…”

“…In first experiments, we characterized the interaction of Hb with enterobacterial LPS and lipid A, and found that the increase of the LPS-induced cytokine induction was due to an enhanced adoption of the 'endotoxic conformation' of the lipid A component of LPS [10]. Thus, we found, that cross-linked Hb is able to convert an inactive pentaacylated having a lamellar conformation lipid A into an endotoxically active cubic conformation [11]. There is evidence that the enhancement of the biological activity is also associated with a Hb-induced disaggregation of LPS [11,12].…”

“…In previously published investigations, the increase in the LPS-induced biological responses caused by Hb was tested in serum-containing systems [10,11]. To exclude the influence of Fig.…”

Monocyte and Macrophage Activation by Lipoteichoic Acid Is Independent of Alanine and Is Potentiated by Hemoglobin

“…Our results suggest that the enhancement of stimulation appears to be due to the formation of complexes between LTA and Hb, but more work will be required to determine the precise nature of this interaction. Hb has been previously shown to form complexes with LPS and to augment its cytokine-stimulating activity (43)(44)(45)(46)(47). The magnitude of enhancement of the activity of LTA appears to be generally greater than that observed for LPS, although further studies will be required to substantiate this point.…”

“…The magnitude of enhancement of the activity of LTA appears to be generally greater than that observed for LPS, although further studies will be required to substantiate this point. For example, at a 1:40 molar ratio of LPS:Hb, there was only a 4-fold increase in TNF-␣ production by human blood cells (47), while at a 1:40 molar ratio of LTA:Hb there was a 40-fold increase in IL-6 secretion by murine macrophages (Fig. 5).…”