Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins

Abstract

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure α-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.

Delineation of Lipopolysaccharide (LPS)-binding Sites on Hemoglobin

“…␣ and ␤ Subunits of Human Hb Harbor LPS Binding SitesPrevious studies have shown that tetrameric Hb from various species, as well as the ␣ subunit of sheep Hb, can bind bacterial LPS (18). Here, we examined whether the isolated subunits of human Hb could also bind LPS.…”

“…The invertebrate respiratory protein, hemocyanin, which is a functional homologue of Hb also binds LPS (12)(13)(14), indicating the evolutionary significance of such interactions. Previous studies have characterized the interactions of Hb and LPS through biophysical methods such as isothermal titration calorimetry and Fouriertransform infrared spectroscopy (17,18), which suggested that Hb intercalates into and alters the aggregate structure of LPS polymers, transitioning it from a relatively dormant multilamellar structure to a bioactive cubic structure. It has been shown that Hb interacts predominantly with the acyl chains and phosphate moieties of lipid A (20).…”

Hemoglobin Enhances the Biological Activity of Synthetic and Natural Bacterial (Endotoxic) Virulence Factors: A General Principle

“…Previously it was shown that Hb causes a disaggregation of LPS Re structures [17]. To continue the examination of this phenomenon we applied freeze-fracture electron microscopy to examine whether this represents a general behaviour.…”

“…The measurements were done as recently described [17]. Briefly, a small amount of the samples was sandwiched between two copper profiles as used for the double-replica technique and frozen by plunging the sandwiches immediately into liquefied ethane-propane-mixture cooled in liquid nitrogen.…”

A Rabbit Vaginal Cell-Derived Antimicrobial Peptide, RVFHbαP, Blocks Lipopolysaccharide-Mediated Inflammation in Human Vaginal CellsIn Vitro

“…Amino acid sequence homology of RVFHb␣P was used for the selection of the template. Two main criteria were included during the selection of template: (i) the selected protein should possess an authenticated crystal structure and (ii) the template must demonstrate maximum homology with the RVFHb␣P amino acid sequence (14). After selecting the template based on the above criteria, its crystal structure was used to model the structure of RVFHb␣P.…”