A novel fluorogenic substrate for the measurement of endothelial lipase activity

Abstract

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

Fig. 1.

Structures of the fluorescent substrates used. A: Chemical structure of PLA₁-specific substrate (N-{[6-(2,4-dinitrophenyl) amino] hexanoyl}-1-{4,4-difluoro-5,7-dimethyl-4-bora-3a,-4a-diaza-s-indacene-3-pentanoyl}-2-hexyl-sn-glycero-3-phosphoethanolamine; mw = 849). B: Chemical structure of PED6 substrate (N-{[6-(2,4-dinitrophenyl) amino]hexanoyl}-2-[4,4-difluoro-5,7-dimethyl-4-bora-3a,-4a-diaza-s-indacene-3-pentanoyl}-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine; mw = 1136). Positions of the sn-1 and sn-2 cleavage sites are indicated for both substrates, and the BODIPY fluor and dinotrophenyl quencher is indicated on the PLA₁ substrate.

Fig. 2.

PLA₁ substrate cleavage products and the mechanism of catalysis. A: Reaction shows how the PLA₁ substrate is hydrolyzed into its two cleavage products by a PLA₁ enzyme such as EL. Molecular weights (MW) of the substrate and predicted products are shown. B: The mechanism by which EL catalyzes the cleavage of the ester bond via an sn-1 reaction is shown. The amino acids (Ser169, Asp 193, and His274) of the catalytic triad reported to be at the active site of murine EL are shown. P1, P2, products 1 and 2.

Fig. 3.

Cleavage of the PLA₁ substrate by purified mouse EL as a function of time and confirmation of the products by LC/MS are shown. Purified mouse EL (200 nM) was incubated with the PLA₁ substrate (10 µM) and monitored for cleavage as a function of time. A: Chromatograms for the 10, 30, 60, and 90 min samples: inset 1, LC/MS chromatogram (h/z 300–350) of the 60 min time point; inset 2, corresponding mass (M) signature of the 3.28 min peak from inset 1; inset 3, LC/MS chromatogram (h/z 800–900) of the 60 min time point; inset 4, corresponding mass signature of the 4.6 min peak from inset 3. B: Quantitation of the PLA₁ substrate turnover as a function time monitored by (ultraviolet [UV]) A₅₀₅ or by MS. C: A Coomassie blue-stained NuPage bis-Tris 4-12%-gradient gel showing purified mouse EL; (A) Full-length mouse EL with cleaved signal sequence amino acids (21–482); (B) Proprotein convertase cleaved mouse EL, N-terminal fragment (amino acids 21–330); (C) Proprotein convertase cleaved mouse EL, C-terminal fragment (amino acids 331–482). Bands were confirmed by N-terminal sequencing.

Fig. 4.

PLA₁ and PED6 substrate turnover by purified LPL and bee venom PLA₂. Comparison demonstrates substrate preferences between purified bovine LPL and bee venom PLA₂, using either the PLA₁ or PED6 substrate. Standard 90 min assays were run with increasing substrate concentrations. This triplicate assay represents one of two different experiments yielding identical results.

Fig. 5.

PED6 substrate versus PLA₁ substrate turnover by EL, HL and LPL in stably expressing HEK293 cell lines. A and B: Cells stably expressing either murine EL, hHL or hLPL were assayed using either the PED6 (A) or PLA₁ (B) substrate. For both substrates, 30 min cell-based assays were run with increasing substrate concentrations. The level of expression for the three lipases in each cell line was comparable, based on qualitative Western blotting (data not shown). These triplicate assays represent one of three different experiments yielding identical results.

Fig. 6.

Ebelactone B inhibition of EL, HL, and LPL in cell-based assays. Cell-based assays with increasing PLA₁ substrate concentrations, using HEK293 untransfected control cells (A) or transfected HEK293 expressing either murine EL (B), hHL (C), or hLPL (D) were run in the absence or presence of the nonselective esterase inhibitor ebelactone B (10 µM). These assays, run in triplicate for 30 min, represent one of three different experiments yielding identical results.

Fig. 7.

PLA₁ substrate turnover by HUVECs is inhibited by ebelactone B. Cell-based assays of HUVECs were run for 120 min, using 2.5 µM PLA₁ substrate and increasing concentrations of ebelactone B. This triplicate assay represents one of two different experiments yielding identical results.