Detection of Lipopolysaccharide(LPS)-Binding Membrane Proteins by Immuno-Coprecipitation with LPS and Anti-LPS Antibodies

In this study we describe a general method for the detection and characterization of endotoxin-(lipo-polysaccharide, LPS)-binding membrane proteins. In the past, experimental procedures to detect LPS-binding sites on cells were generally performed with chemically modified LPS derivates. Since anymodification of a ligand may lead to a modification of its binding characteristics, the results of thosestudies are controversial.In our assay, cell membrane preparations are treated with free lipid A, the endotoxic center of LPS,in the presence of normal human serum. After binding of lipid A, membrane proteins are solubilized bymild detergent treatment without disruption of the lipid A-protein complexes. Addition of anti-(lipid A)mAbs and subsequent adding of protein A agarose lead to the precipitation of complexes of lipid A andits binding proteins. By SDS/PAGE and western blot, these precipitates can be screened for the presenceof LPS/lipid A-binding proteins. We describe the use of this method for the immuno-coprecipitation oflipid A (or LPS) with an 80-kDa LPS-binding membrane protein (LMP80), which we have previouslyidentified on several human cells. In addition, CD14, the well-known functional LPS receptor on mono-cytes and macrophages, can be detected.By means of this immuno-coprecipitation approach we could demonstrate binding of either purifiedLPS preparations or synthetic lipid A to these LPS/lipid A-binding membrane proteins at physiologicalpH under conditions in which the proteins are in their natural membranous environment.Keywords: lipopolysaccharide; lipopolysaccharide-binding protein; CD14; anti-lipopolysaccharide anti-body; 80-kDa lipopolysaccharide-binding membrane protein (LMP80).Lipopolysaccharides (LPS, endotoxin) constitute the major biological activity and, therefore, a putative LPS-binding mole-component of the outer membrane of gram-negative bacteria. cule should also bind lipid A. In the immuno-coprecipitationLPS are responsible for many pathophysiological effects ob- assay we incubated cell membrane preparations, which containserved during gram-negative septic episodes [1, 2]. The LPS of the proteins in their native conformation, with lipid A. Afterall families of gram-negative bacteria contains the highly con- binding of lipid A to the LPS-binding molecules, the cell mem-served lipid part termed lipid A, which is able to mimic the branes were washed several times and solubilized by mild de-biological effects of LPS [1, 3]. Thus, lipid A represents the tergent lysis without disruption of the lipid A-protein complexes.endotoxic center of LPS. These complexes were precipitated by addition of anti-(lipid A)Many experimental procedures for the detection of the cellu- mAbs together with protein A agarose beads. After some wash-lar targets of LPS in host cell membranes have been carried out ing steps the precipitates were eluted by adding SDS sampleunder non physiological circumstances: Either the LPS prepara- buffer (62.5 mM Tris, 80 mM dithiothreitol,10% glycerol, 2%tion was chemically modified by labeling with or substitution SDS, 0.002% Bromophenol blue, pH 6.8) and separated by SDS/for a cross-linker, or the proteins under investigation were not PAGE. Specific proteins were detected by an immunoblot tech-in their physiological conformation after SDS/PAGE and west- nique on nitrocellulose membrane. The immuno-coprecipitationern blot techniques. Modification of a receptor molecule or its method not only leads to the detection of LPS-binding proteins,ligands for binding studies is critical, as the modification may it also permits the enrichment of those molecules from cellhave a strong influence on the binding behaviour. Therefore, the membrane preparations.physiological relevance of many molecules found by the use of Here, we demonstrate the successful use of this method bythose techniques remains controversial. the identification of lipid A binding to an 80-kDa LPS-bindingTo obtain a more physiological detection system for LPS- membrane protein (LMP80), which was previously described bybinding molecules we developed an immuno-coprecipitation our group [4] as well as to the LPS receptor CD14. Furthermore,assay with free lipid A and anti-(lipid A) antibodies. Lipid A we examined the binding of LPS as well as synthetic oligoacylwas used, because it is the minimal structure of LPS with full lipid A partial structures to LMP80 [527].