Synthesis of [3H]-Labeled Bioactive Lipid A Analogs and Their Use for Detection of Lipid A-Binding Proteins on Murine Macrophages

Abstract

Abstract Both endotoxic and antagonistic [3H]-labeled 2-(phosphonooxy)ethyl (PE) analogs of lipid A were synthesized with high purity and high specific radioactivity. Lipid A-binding proteins were detected by using the endotoxic analog of hexaacyl Escherichia coli-type designated [3H] PE-506. The plasma membrane fractions from peritoneal macrophages derived from LPS-responder C3H/HeN mice and LPS-hyporesponder C3H/HeJ mice were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then incubated with the [3H] PE-506. Several [3H] PE-506 binding proteins were detected in both C3H/HeN and C3H/HeJ macrophages. Unlabeled hexaacyl lipid A inhibited the interaction between [3H] PE-506 and these proteins. The result suggests that there exist multiple binding sites for lipid A on macrophages. LPS-induced change in the profile of the cell surface lipid A binding proteins was observed in C3H/HeN macrophages, but not in C3H/HeJ macrophages, by preincubation of macrophages with LPS.