Activation and inhibition of Limulus amebocyte lysate coagulation by chemically defined substructures of lipid A

Recent work with lipid mutants of Escherichia coli and Salmonella typhimurium has helped to elucidate the correct structure of lipid A and has suggested a biosynthetic pathway. Precursor molecules include diacylglucosamine 1-phosphates and tetraacyl disaccharide bis-phosphates. The activities of several of these compounds and of their derivatives were measured by Limulus amebocyte lysate (LAL) assay. We report that (i) both mono- and disaccharide precursors of lipid A activate LAL, (ii) two acyl chains on the monosaccharide subunit of lipid A are necessary for activation of LAL, and (iii) the monosaccharide, 2-monoacylglucosamine 1-phosphate can competitively inhibit LAL activation by diacyl monosaccharide lipid A precursors. However, 2-monoacylglucosamine 1-phosphate did not inhibit endotoxin activation of LAL. One unanticipated finding was that the activities of the monosaccharides were reduced upon storage even though their covalent structures were unchanged. Perhaps this is due to alterations in physical state. Thus, these lipid A precursors and derivatives offer some insight into the structural features required for activation of the LAL assay and may in the future provide derivatives which are competitive inhibitors of endotoxin.