Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Abstract

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum. Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum. Endothelial lipase (EL) belongs to the vascular heparan sulfate-associated family of lipases (for review, see Refs. 1Jaye M. Krawiec J. Endothelial lipase and HDL metabolism. Curr. Opin. Lipidol. 2004; 15: 183-189Crossref PubMed Scopus (41) Google Scholar, 2Badellino K.O. Rader D.J. The role of endothelial lipase in high-density lipoprotein metabolism. Curr. Opin. Cardiol. 2004; 19: 392-395Google Scholar, 3Broedl U.C. Jin W. Rader D.J. Endothelial lipase: a modulator of lipoprotein metabolism upregulated by inflammation. Trends Cardiovasc. Med. 2004; 14: 202-206Google Scholar). Members of this family include LPL, HL, and pancreatic lipase. Like LPL and HL, EL is believed to function mainly in the plasma compartment, where it hydrolyzes phospholipids, particularly in HDL. Although predominantly a phospholipase, EL also hydrolyzes triglycerides in lipoprotein particles (4McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase. J. Lipid Res. 2002; 43: 921-929Google Scholar). Genetic deletion of EL, or administration of inhibitory antibodies to EL, resulted in significant increases in high density lipoprotein cholesterol (HDL-C) levels in mice (5Ishida T. Choi S. Kundu R.K. Hirata K. Rubin E.M. Cooper A.D. Quertermous T. Endothelial lipase is a major determinant of HDL level. J. Clin. Invest. 2003; 111: 347-355Google Scholar, 6Ma K. Cilingiroglu M. Otvos J.D. Ballantyne C.M. Marian A.J. Chan L. Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism. Proc. Natl. Acad. Sci. USA. 2003; 100: 2748-2753Google Scholar, 7Jin W. Millar J.S. Broedl U. Glick J.M. Rader D.J. Inhibition of endothelial lipase causes increased HDL cholesterol levels in vivo. J. Clin. Invest. 2003; 111: 357-362Google Scholar). The significance of this increase in HDL-C can be seen in one study in which EL and apolipoprotein E (apoE) double deficient mice had a considerable improvement in atherosclerosis, as measured b