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

Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1’s acidic domain preserves LPL structure and activity, we crystallized an LPL–GPIHBP1 complex and solved its structure. GPIHBP1’s LU domain binds to LPL’s C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1’s acidic domain was not defined in the electron density map but was positioned to interact with LPL’s large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL–GPIHBP1 structure provides insights into mutations causing chylomicronemia.

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A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806774115 ◽

2018 ◽

Vol 115 (26) ◽

pp. E6020-E6029 ◽

Cited By ~ 19

Author(s):

Kristian K. Kristensen ◽

Søren Roi Midtgaard ◽

Simon Mysling ◽

Oleg Kovrov ◽

Lars Bo Hansen ◽

...

Keyword(s):

Endothelial Cells ◽

Lipoprotein Lipase ◽

Catalytic Domain ◽

Intrinsically Disordered ◽

Severe Hypertriglyceridemia ◽

Biophysical Studies ◽

Close Proximity ◽

Key Factor ◽

Capillary Endothelial Cells ◽

Structure And Activity

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL’s catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1’s IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1–LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1–LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1’s IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1’s ability to preserve LPL structure and activity.

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Assessing the Role of the Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) Three-finger Domain in Binding Lipoprotein Lipase

Journal of Biological Chemistry ◽

10.1074/jbc.m111.242024 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19735-19743 ◽

Cited By ~ 40

Author(s):

Anne P. Beigneux ◽

Brandon S. J. Davies ◽

Shelly Tat ◽

Jenny Chen ◽

Peter Gin ◽

...

Keyword(s):

High Density Lipoprotein ◽

Lipoprotein Lipase ◽

Binding Protein ◽

Density Lipoprotein ◽

High Density ◽

Cell Protein ◽

Apical Surface ◽

Amino Terminal ◽

Acidic Domain ◽

Lipoprotein Binding

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.

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Severe hypertriglyceridemia due to two novel loss-of-function lipoprotein lipase gene mutations (C310R/E396V) in a Chinese family associated with recurrent acute pancreatitis

Oncotarget ◽

10.18632/oncotarget.17762 ◽

2017 ◽

Vol 8 (29) ◽

pp. 47741-47754 ◽

Cited By ~ 4

Author(s):

Yu Lun ◽

Xiaofang Sun ◽

Ping Wang ◽

Jingwei Chi ◽

Xu Hou ◽

...

Keyword(s):

Acute Pancreatitis ◽

Lipoprotein Lipase ◽

Gene Mutations ◽

Chinese Family ◽

Loss Of Function ◽

Lipase Gene ◽

Recurrent Acute Pancreatitis ◽

Lipoprotein Lipase Gene ◽

Severe Hypertriglyceridemia

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The angiopoietin-like protein ANGPTL4 catalyzes unfolding of the hydrolase domain in lipoprotein lipase and the endothelial membrane protein GPIHBP1 counteracts this unfolding

eLife ◽

10.7554/elife.20958 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 33

Author(s):

Simon Mysling ◽

Kristian Kølby Kristensen ◽

Mikael Larsson ◽

Oleg Kovrov ◽

André Bensadouen ◽

...

Keyword(s):

Lipoprotein Lipase ◽

Molecular Basis ◽

Plasma Triglyceride ◽

Lower Plasma ◽

Genetic Studies ◽

Molecular Defects ◽

Intrinsically Disordered ◽

Acidic Domain ◽

Physiological Relevance ◽

Α Helix

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

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The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

eLife ◽

10.7554/elife.12095 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Simon Mysling ◽

Kristian Kølby Kristensen ◽

Mikael Larsson ◽

Anne P Beigneux ◽

Henrik Gårdsvoll ◽

...

Keyword(s):

Membrane Protein ◽

Lipoprotein Lipase ◽

Catalytic Domain ◽

Exchange Mass Spectrometry ◽

Intrinsically Disordered ◽

Acidic Domain ◽

Terminal Domain ◽

Capillary Endothelial Cells ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

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Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920202117 ◽

2020 ◽

Vol 117 (8) ◽

pp. 4337-4346 ◽

Cited By ~ 7

Author(s):

Kristian K. Kristensen ◽

Katrine Zinck Leth-Espensen ◽

Haydyn D. T. Mertens ◽

Gabriel Birrane ◽

Muthuraman Meiyappan ◽

...

Keyword(s):

Lipoprotein Lipase ◽

Lipid Binding ◽

Carboxyl Terminus ◽

Specific Monoclonal Antibody ◽

Triglyceride Hydrolysis ◽

Severe Hypertriglyceridemia ◽

Capillary Endothelial Cells ◽

Hydrolysis Of ◽

Insight Into ◽

Binding Loop

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

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