A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time

Abstract

The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceride-derived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. Physiologically active LPL is normally bound to the endothelial cell protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), which transports LPL across endothelial cells, anchors LPL to the vascular wall, and stabilizes LPL activity. Disruption of LPL-GPIHBP1 binding significantly alters triglyceride metabolism and lipid partitioning. In this study, we modified the NanoLuc® Binary Technology split-luciferase system to develop a novel assay that monitors the binding of LPL to GPIHBP1 on endothelial cells in real time. We validated the specificity and sensitivity of the assay using endothelial lipase and a mutant version of LPL and found that this assay reliably and specifically detected the interaction between LPL and GPIHBP1. We then interrogated various endogenous and exogenous inhibitors of LPL-mediated lipolysis for their ability to disrupt the binding of LPL to GPIHBP1. We found that angiopoietin-like (ANGPTL)4 and ANGPTL3-ANGPTL8 complexes disrupted the interactions of LPL and GPIHBP1, whereas the exogenous LPL blockers we tested (tyloxapol, poloxamer-407, and tetrahydrolipstatin) did not. We also found that chylomicrons could dissociate LPL from GPIHBP1 and found evidence that this dissociation was mediated in part by the fatty acids produced by lipolysis. These results demonstrate the ability of this assay to monitor LPL-GPIHBP1 binding and to probe how various agents influence this important complex.

Keywords: NanoLuc® Binary Technology; chylomicrons; endothelial cell; glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1; lipolysis; lipoprotein metabolism; triglyceride.

Fig. 1.

Association of largeBiT-LPL with smallBiT-GPIHBP1. A: Schematic of the NanoBiT LPL binding assay. B: SmallBiT-GPIHBP1-expressing RHMVECs were grown to confluence. Luminescent substrate (Nano-Glo® live cell substrate) was added to cells and luminescence was read every minute. After 6 min (dotted line), the indicated concentrations of largeBiT-LPL or largeBiT-EL were added to each well and luminescence continued to be measured each minute for an additional 30 min. Points represent mean ± 95% CI of four independent experiments (n = 6 per group per experiment). C: Western blot of the LargeBiT LPL (LPL-LgBiT)- and LargeBiT-EL (EL-LgBiT)-conditioned media used in B. D: Western blot of the LargeBiT LPL (LPL)- and LargeBiT-LPL C445Y (mutLPL)-conditioned media used in E. E: SmallBiT-GPIHBP1-expressing RHMVECs were grown to confluence. Luminescent substrate was added to cells and luminescence was read every minute. After 6 min (dotted line), the indicated concentrations of largeBiT-LPL (LPL) or largeBiT-LPL C445Y (mutLPL) were added to each well and luminescence continued to be measured each minute for an additional 30 min. Points represent mean ± 95% CI of three independent experiments (n = 6 per group per experiment).

Fig. 2.

Disruption of LPL-GPIHBP1 binding by heparin. A: Schematic of the NanoBiT LPL dissociation assay. B: Western blot of lysates of smallBiT-GPIHBP1-expressing RHMVECs incubated with LargeBiT-LPL for 3.5 h at 4°C, washed, and then treated with or without 100 U/ml heparin for 30 min. C, D: SmallBiT-GPIHBP1-expressing endothelial cells were incubated with LargeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to cells and luminescence read every 3 min for 15 min. After 15 min 100 U/ml heparin (+heparin) or water (–heparin) were added to the samples and luminescence was read every 3 min for an additional 15 min. C: Luminescent signal over time (maximum signal for each sample set to 100%). D: Luminescence signal over time normalized to the –heparin control at each time point. Points represent mean ± 95% CI of three independent experiments (n = 3–6 per group per experiment).

Fig. 3.

Disruption of LPL-GPIHBP1 binding by ANGPTL proteins. A: RHMVECs expressing smallBiT-GPIHBP1 were incubated with largeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to cells and luminescence read every 3 min for 15 min. After 15 min (dotted line), the indicated ANGPTL proteins were added and luminescence continued to be measured every 3 min for 45 min. Points represent mean ± 95% CI of luminescent signal over time normalized to the “LPL only” control at each time point. Data represent seven independent experiments with three biological replicates per group. B: Western blot of cell lysates from cells incubated with largeBiT-LPL for 3.5 h at 4°C, washed, and then treated with ANGPTL4, ANGPTL3, ANGPTL8, or heparin for 30 min at 37°C. Bands show LPL (using an antibody against the FLAG tag), GPIHBP1, and actin for biological triplicates. C: LPL activity of LPL bound to smallBiT-GPIHBP1-expressing RHMVECs washed and treated with ANGPTL4 (A4), ANGPTL3 (A3), ANGPTL8 (A8), or ANGPTL3 and ANGPTL8 (A3/8) for 30 min at 37°C. After incubation, cells were washed, and LPL was released from the cells with heparin before performing LPL activity assays. Bars represent normalized LPL activity (mean ± 95% CI of three independent experiments; n = 3 per experiment). D: Luminescence of smallBiT-GPIHBP1-expressing RHMVECs incubated with largeBiT-LPL. After washing off unbound LPL, luminescence was measured for 15 min at 3 min intervals. The indicated ANGPTL proteins were added (first dotted line) and luminescence was measured for an additional 30 min. Cells were then washed, fresh substrate was added (second dotted line), and luminescence was measured for another 15 min. Points represent luminescent signal normalized to the LPL only control at each time point (mean ± 95% CI of four independent experiments; n = 3 per group per experiment).

Fig. 4.

Effect of THL on GPIHBP1-LPL binding. A: RHMVECs expressing smallBiT-GPIHBP1 were incubated with largeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to the cells, and luminescence was read every 3 min for 15 min. After 15 min (dotted line), 0–80 μM of THL were added and luminescence continued to be measured every 3 min for 45 min. Points represent mean ± 95% CI of luminescent signal over time normalized to the vehicle (ethanol). Data represent three independent experiments each with three biological replicates per group. B: Western blot of cell lysates from cells incubated with largeBiT-LPL for 3.5 h at 4°C, washed, and then treated with 80 μM of THL or 100 U/ml heparin for 30 min. Bands show LPL (using an antibody against the FLAG tag), GPIHBP1, and actin for biological triplicates. C, D: LPL activity of largeBiT-LPL bound to smallBiT-GPIHBP1-expressing RHMVECs (C) or FLAG-LPL bound to S tag-GPIHBP1 (D) after treatment with 80 μM of THL for 30 min at 37°C. After incubation, the cells were washed, and LPL was released from the cells with heparin before performing LPL activity assays. Bars represent normalized LPL activity (mean ± 95% CI of three independent experiments; n = 6 per experiment).

Fig. 5.

Effect of chylomicrons and fatty acids on GPIHBP1-LPL binding. RHMVECs expressing smBiT-GPIHBP1 were incubated with LargeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to cells and luminescence read every 3 min for 12–15 min. A: After 15 min (dotted line), chylomicrons (chylo) (1–10 μg/ml by protein) were added and luminescence continued to be measured for 45 min. ANGPTL4 (90 ng/ml) was also added as a control. B: After 12 min, chylomicrons or heparin were added (first dotted line) at the indicated concentrations and luminescence was measured for an additional 12 min. Cells were then washed, fresh substrate was added (second dotted line), and luminescence was measured for another 15 min. C: After 15 min (dotted line), sodium oleate (0.375–3 mM) was added and luminescence continued to be measured for 45 min. For all panels, points represent luminescent signal over time normalized to control at each time point (mean ± 95% CI of three independent experiments; n = 3–6 per group per experiment).

Fig. 6.

Effect of THL on chylomicron-LPL interactions. A: RHMVECs expressing smBiT-GPIHBP1 were incubated with LargeBiT-LPL for 2 h at 4°C and washed. Cells were then treated with 80 μM of THL or vehicle for 30 min at 37°C. After washing, luminescent substrate was added to cells and luminescence read every 3 min for 12 min. After 12 min, chylomicrons (Chylo) (0–20 μg/ml by protein) were added and luminescence continued to be measured for 33 min. Points represent mean ± 95% CI of luminescent signal over time normalized to control at each time point. Data represent three independent experiments, each with 3–6 biological replicates per group. B: Immunofluorescence showing binding of LPL and chylomicrons to smallBiT-GPIHBP1-expressing RHMVECs. RHMVECs were incubated with largeBiT-LPL for 3 h at 4°C. After washing away unbound LPL, cells were incubated with or without THL (80 μM) for 30 min at 37°C. Cells were then washed and incubated with or without fluorescently labeled chylomicrons (green) for 30 min at 37°C. Cells were then stained for LPL (red) using an antibody against the FLAG tag and with DAPI (blue).

Fig. 7.

Effect of tyloxapol and P-407 on GPIHBP1-LPL binding and LPL activity. A, B: RHMVECs expressing smallBiT-GPIHBP1 were incubated with largeBiT-LPL for 2 h at 4°C and washed. Luminescent substrate was added to cells and luminescence read every 3 min for 15 min. After 15 min (dotted line), 150–600 μg/ml of tyloxapol (tylox) (A) or 2–8 mg/ml of P-407 (B) were added and luminescence continued to be measured for 45 min. Points represent mean ± 95% CI of luminescent signal over time normalized to the control at each time point. Data represent three independent experiments, each with three biological triplicates per group. C, D: Western blot of cell lysates from cells incubated with LargeBiT-LPL for 3.5 h at 4°C, washed, and then treated with 8 mg/ml tyloxapol (C) or 8 mg/ml P-407 (D) for 30 min at 37°C. Bands show LPL (using an antibody against the FLAG tag), GPIHBP1, and actin for biological triplicates. E, F: Luminescence of smallBiT-GPIHBP1-expressing RHMVECs incubated with LgBiT-LPL. After washing off unbound LPL, luminescence was measured for 12 min (3 min intervals). Tyloxapol (E) or P-407 (F) was added (first dotted line) at the indicated concentrations and luminescence was measured for an additional 12 min. Cells were then washed, fresh substrate was added (second dotted line), and luminescence was measured for another 15 min. Points represent luminescent signal over time normalized to the LPL only control at each time point (mean ± 95% CI of three independent experiments; n = 3–6 per group per experiment). G–J: LPL activity of LPL bound to smallBiT-GPIHBP1-expressing RHMVECs washed and treated with 0 (control), 2, or 8 mg/ml tyloxapol (G, H) or 0 (control), 2, or 8 mg/ml poloxamer (I, J) for 30 min at 37°C. For G and I, heparin was added directly to the reaction to release LPL. For H and J, cells were washed extensively and then LPL was released from the cells with heparin. In all cases, LPL activity assays were performed on heparin-released LPL. Bars represent normalized LPL activity (mean ± 95% CI of three independent experiments; n = 3–6 per experiment).

Fig. 8.

Effect of tyloxapol on binding of chylomicrons to LPL. A: Chylomicrons (20 μg/ml) were mixed with 2 or 8 mg/ml tyloxapol (tylox) and then incubated with largeBiT-LPL bound to smallBiT-GPIHBP1-expressing RHMVECs at 37°C. After 30 min, cells were washed, substrate was added, and luminescence was read. Bars represent luminescent signal (mean ± 95% CI of four independent experiments; n = 4 per group per experiment) normalized to the no chylomicron no tyloxapol control. B: Immunofluorescence showing binding of LPL and chylomicrons (chylos) to smallBiT-GPIHBP1-expressing RHMVECs. RHMVECs were incubated with largeBiT-LPL for 3.5 h at 4°C. After washing away unbound LPL, cells were incubated with or without with 20 µg/ml fluorescently labeled chylomicrons (green) for 30 min at 37°C, then with or without 8 mg/ml tyloxapol for 30 min at 37°C. Cells were then stained for LPL (red) using an antibody against the Flag tag and with DAPI (blue). C: Distribution of chylomicron diameters before (baseline) and after 30 min treatment with 8 mg/ml tyloxapol as measured by dynamic laser light scattering. Untreated chylomicrons stored at 4°C for 30 min after baseline reading were also analyzed (30 min untreated). Lines represent mean ± range (shaded area) particle distribution of three different reads.