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

Shwetha K Shetty,Rosemary L Walzem,Brandon S.J Davies

doi:10.1194/jlr.d119000388

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.

Highlights

The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceridederived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues
Delivery of triglyceride-derived fatty acids to peripheral tissues such as heart, adipose tissue, and skeletal muscle is facilitated by the action of LPL, an extracellular triglyceride lipase that hydrolyzes the triglycerides of triglyceride-rich lipoproteins (i.e., VLDL and chylomicrons)
We asked whether the binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) on the surface of endothelial cells could be detected in real time by monitoring luminescence (Fig. 1A)

Summary

Introduction

The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceridederived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. We 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.—Shetty, S. Deficiency in ANGPTL3, ANGPTL4, ANGPTL8, or Apo-C3 results in decreased plasma triglyceride levels and, in the case of ANGPTL3, ANGPTL4, and Apo-C3, appears to protect against cardiovascular disease [13,14,15,16,17,18,19,20,21,22] Therapies targeting these endogenous inhibitors have gained traction in preclinical studies and clinical trials [13,14,15, 23,24,25,26,27,28,29].

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Conclusion