Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade

Abstract

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (pl-6)-~-Glocosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (j1-6)-~-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity.Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.A series of compounds corresponding in structure to the lipid A portions of endotoxic lipopolysaccharides of Enterobacteriaceae, their analogs and their structural components have been synthesized [l -71. Endotoxic and immunobiological activities of these synthetic compounds have been extensively studied in comparison with those of reference lipopolysaccharides and lipid A preparations of bacterial origin by a few groups including ours [8-231. A synthetic counterpart (LA-1 5-PP) of lipid A from an Escherichiu coli Re mutant exhibited full endotoxic and immunobiological activities identical to those of a reference bacterial product [lo, 15,191. The bioactivities of a synthetic compound...

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities

“…For an understanding, the structural requirements of the activation of the clotting cascade must be considered. By using different synthetic lipid A and part structures, Takada et al [31] have shown that the monophosphoryl 4'-phosphate lipid A has the same activity as bisphosphoryl lipid A, whereas the monophosphoryl 1-phosphate lipid A has strongly reduced activity. We have found that the interaction of LPS and lipid A with hemoglobin [32] or albumin [33] led to a strong or slight increase, respectively, of the cytokine production in mononuclear cells, but was not correlated with the results in the Limulus test, which exhibited somehow non-systematic variations.…”

Sensing Gram-Negative Bacterial Lipopolysaccharides: a Human Disease Determinant?

“…The most widely employed test for the presence of endotoxin in fluids is based on LPS sensing by the clotting system of the horseshoe crab. This assay is not specific for hexaacyl LPS (132,133). LPS recognition by the horseshoe crab likely evolved as a defense from aquatic bacteria; the assay's ability to recognize widely diverse lipid A structures enhances its ability to detect environmental contamination of biologicals, but it also may account for some of the problems observed when the test has been used to detect endotoxemia and gram-negative bacteremia in human patients (the correlations between disease severity, gram-negative bacteremia and endotoxemia have generally been poor) (57).…”

Neutralizing and cross-reactive antibodies against enterobacterial lipopolysaccharide

“…Degradation by chemical means showed that upon treatment with mild aqueous acid, a precipitate could be obtained which was termed lipid A and represents the lipid anchor of the LPS molecule in the outer membrane (Westphal and Lu¨deritz, 1954;Imoto et al, 1985), and which was later on identified as the endotoxic principle of LPS (Lu¨deritz et al, 1984;Galanos et al, 1985;Kotani et al, 1985). A detailed investigation of structure-activity relationships (Kotani et al, 1984;Takada et al, 1988;Rietschel et al, 1994), including a biophysical characterization of biologically active and inactive lipid A revealed structural parameters which influence the biological activities of LPS (Rietschel et al, 1990;Seydel et al, 1999).…”