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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.

SUBMITTER: Shetty SK

PROVIDER: S-EPMC7112140 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Chylomicronemia mutations yield new insights into interactions between lipoprotein lipase and GPIHBP1.

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Structure of lipoprotein lipase in complex with GPIHBP1.

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GPIHBP1 missense mutations often cause multimerization of GPIHBP1 and thereby prevent lipoprotein lipase binding.

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Structure of the lipoprotein lipase-GPIHBP1 complex that mediates plasma triglyceride hydrolysis.

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| S-EPMC6358717 | biostudies-literature

A negatively charged cluster in the disordered acidic domain of GPIHBP1 provides selectivity in the interaction with lipoprotein lipase.

Project description:GPIHBP1 is a membrane protein of endothelial cells that transports lipoprotein lipase (LPL), the key enzyme in plasma triglyceride metabolism, from the interstitial space to its site of action on the capillary lumen. An intrinsically disordered highly negatively charged N-terminal domain of GPIHBP1 contributes to the interaction with LPL. In this work, we investigated whether the plethora of heparin-binding proteins with positively charged regions found in human plasma affect this interaction. We also wanted to know whether the role of the N-terminal domain is purely non-specific and supportive for the interaction between LPL and full-length GPIHBP1, or whether it participates in the specific recognition mechanism. Using surface plasmon resonance, affinity chromatography, and FRET, we were unable to identify any plasma component, besides LPL, that bound the N-terminus with detectable affinity or affected its interaction with LPL. By examining different synthetic peptides, we show that the high affinity of the LPL/N-terminal domain interaction is ensured by at least ten negatively charged residues, among which at least six must sequentially arranged. We conclude that the association of LPL with the N-terminal domain of GPIHBP1 is highly specific and human plasma does not contain components that significantly affect this complex.

| S-EPMC11344153 | biostudies-literature

Chylomicronemia with low postheparin lipoprotein lipase levels in the setting of GPIHBP1 defects.

Project description:Recent studies in mice have established that an endothelial cell protein, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), is essential for the lipolytic processing of triglyceride-rich lipoproteins.We report the discovery of a homozygous missense mutation in GPIHBP1 in a young boy with severe chylomicronemia. The mutation, p.C65Y, replaces a conserved cysteine in the GPIHBP1 lymphocyte antigen 6 domain with a tyrosine and is predicted to perturb protein structure by interfering with the formation of a disulfide bond. Studies with transfected Chinese hamster ovary cells showed that GPIHBP1-C65Y reaches the cell surface but has lost the ability to bind lipoprotein lipase (LPL). When the GPIHBP1-C65Y homozygote was given an intravenous bolus of heparin, only trace amounts of LPL entered the plasma. We also observed very low levels of LPL in the postheparin plasma of a subject with chylomicronemia who was homozygous for a different GPIHBP1 mutation (p.Q115P). When the GPIHBP1-Q115P homozygote was given a 6-hour infusion of heparin, a significant amount of LPL appeared in the plasma, resulting in a fall in the plasma triglyceride levels from 1780 to 120 mg/dL.We identified a novel GPIHBP1 missense mutation (p.C65Y) associated with defective LPL binding in a young boy with severe chylomicronemia. We also show that homozygosity for the C65Y or Q115P mutations is associated with low levels of LPL in the postheparin plasma, demonstrating that GPIHBP1 is important for plasma triglyceride metabolism in humans.

| S-EPMC2858258 | biostudies-literature

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