57] Eukaryotic lipopolysaccharide deacylating enzyme

Abstract

Publisher Summary This chapter discusses the eukaryotic lipopolysaccharide deacylating enzyme. Human neutrophils contain an enzyme—acyloxyacyl hydrolase (AOAH)—that selectively cleaves secondary (acyloxyacyl-linked) acyl chains from the lipid A region of bacterial lipopolysaccharides (LPS). Enzyme(s) with this activity has also been found in human monocytes, macrophages, and vascular endothelial cells, but not in platelets, erythrocytes, or fibroblasts; the activity has also been found in leukocytes from mice, rabbits, cows, pigs, and chickens. The purified human neutrophil enzyme is a glycoprotein that has two disulfide-linked subunits. In vitro, AOAH hydrolyzes LPS acyloxyacyl bonds that have structurally different secondary acyl chains, regardless of the location of the acyloxyacyl linkage on the diglucosamine lipid A backbone; it does not remove the 3-hydroxyacyl chains that are directly linked to the backbone. Recent studies in laboratory indicate that the enzyme also has phospholipase A1, lysophospholipase, and diglyceride lipase activities in vitro. The enzyme may be purified from human neutrophils by a series of standard chromatographic steps or by monoclonal antibody affinity chromatography followed by LPS-agarose affinity chromatography. AOAH activity is measured by quantitating the release of 3H-labeled fatty acids from a biosynthetically double-labeled LPS substrate that contains 3H-labeled acyl chains and 14C-labeled glucosamine.