Aggregation Behavior of an Ultra-Pure Lipopolysaccharide that Stimulates TLR-4 Receptors

The innate immune systems of humans and other animals are activated by lipopolysaccharides (LPS), which are glucosamine-based phospholipids that form the outer leaflet of the outer membranes of Gram-negative bacteria. Activation involves interactions of LPS with the innate immunity-receptor comprised of toll-like receptor 4 in complex with so-called MD-2 protein and accessory proteins, such as CD14 and LPS binding protein. The Lipid Metabolites and Pathways Strategy (LIPID MAPS) Consortium has isolated in large amounts a nearly homogeneous LPS, Kdo2-Lipid A, and demonstrated that it activates macrophages via toll-like receptor 4. The active form of LPS, monomer or aggregate, is controversial. We have therefore examined the aggregation behavior and other physical properties of Kdo2-Lipid A. Differential scanning calorimetry of Kdo2-Lipid A suspensions revealed a gel-to-liquid crystalline phase transition at 36.4°C (Tm). The nominal critical aggregation concentration, determined by dynamic light scattering, was found to be 41.2 ± 1.6 nM below the Tm (25°C), but only 8.1 ± 0.3 nM above the Tm (37°C). The specific molecular volume of Kdo2-Lipid A, obtained by densitometry measurements was found to be 3159 ± 71 Å3 at 25°C, from which the number of molecules in each aggregate was estimated to be 5.8 × 105. The aggregation behavior of Kdo2-Lipid A in the presence of lipoprotein-deficient serum suggests that Re LPS monomers and multimers are the active units for the immune system in the CD14-dependent and -independent pathways, respectively.