Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism that hydrolyses triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. The enzyme is thought to play a role in the development of obesity and atherosclerosis. Defects in LPL are a cause of familial chylomicronemia syndrome (or type I hyperlipoproteinemia) and also of a form of deficiency characterised by hypertriglyceridemia. Familial chylomicronemia is a recessive disorder usually manifesting in childhood. On a normal diet, patients often present with abdominal pain, hepatosplenomegaly, lipemia retinalis, eruptive xanthomata, and massive hypertriglyceridemia, sometimes complicated with acute pancreatitis. Endothelial lipase (encoded by the LIPG gene) regulates the circulating level of high density lipoprotein cholesterol (HDL-C). It can also form a molecular bridge between endothelial cells and lipoproteins or circulating macrophages through interaction with heparan sulfate proteoglycans. This nonenzymatic action can increase cellular lipoprotein uptake and monocyte adhesion and contribute to atherosclerosis. LPL is a secreted glycoprotein that contains five disulfide bonds and requires an endoplasmic reticulum (ER) protein, lipase maturation factor 1 (LMF1), to successfully fold and traffic out of the ER to the Golgi. LPL is sorted into vesicles in an inactive state: helical LPL oligomer. LPL secretion is mediated by Syndecan-1 (SDC1), a heparan sulfate proteoglycan (HSPG). Stored LPL can be secreted into the interstitial space, where it interacts with HSPGs that bind to the multiple heparin binding sites on each LPL molecule . LPL is next bound by glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) and transported into the capillary, where it acts on chylomicrons and very-low-density lipoproteins (VLDLs) to hydrolyze packaged triglycerides and release FFAs. The angiopoietin-like (ANGPTL) family of proteins inhibit LPL in different tissues. Leth-Espensen et al. publish that the intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding. Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase is published by Kumari et al.
27 moreTitle: The GPIHBP1-LPL complex and its role in plasma triglyceride metabolism: Insights into chylomicronemia Jiang S, Ren Z, Yang Y, Liu Q, Zhou S, Xiao Y Ref: Biomed Pharmacother, 169:115874, 2023 : PubMed
GPIHBP1 is a protein found in the endothelial cells of capillaries that is anchored by glycosylphosphatidylinositol and binds to high-density lipoproteins. GPIHBP1 attaches to lipoprotein lipase (LPL), subsequently carrying the enzyme and anchoring it to the capillary lumen. Enabling lipid metabolism is essential for the marginalization of lipoproteins alongside capillaries. Studies underscore the significance of GPIHBP1 in transporting, stabilizing, and aiding in the marginalization of LPL. The intricate interplay between GPIHBP1 and LPL has provided novel insights into chylomicronemia in recent years. Mutations hindering the formation or reducing the efficiency of the GPIHBP1-LPL complex are central to the onset of chylomicronemia. This review delves into the structural nuances of the GPIHBP1-LPL interaction, the consequences of mutations in the complex leading to chylomicronemia, and cutting-edge advancements in chylomicronemia treatment.
The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's alpha/beta-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal alpha-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.
Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.
        
27 lessTitle: The GPIHBP1-LPL complex and its role in plasma triglyceride metabolism: Insights into chylomicronemia Jiang S, Ren Z, Yang Y, Liu Q, Zhou S, Xiao Y Ref: Biomed Pharmacother, 169:115874, 2023 : PubMed
GPIHBP1 is a protein found in the endothelial cells of capillaries that is anchored by glycosylphosphatidylinositol and binds to high-density lipoproteins. GPIHBP1 attaches to lipoprotein lipase (LPL), subsequently carrying the enzyme and anchoring it to the capillary lumen. Enabling lipid metabolism is essential for the marginalization of lipoproteins alongside capillaries. Studies underscore the significance of GPIHBP1 in transporting, stabilizing, and aiding in the marginalization of LPL. The intricate interplay between GPIHBP1 and LPL has provided novel insights into chylomicronemia in recent years. Mutations hindering the formation or reducing the efficiency of the GPIHBP1-LPL complex are central to the onset of chylomicronemia. This review delves into the structural nuances of the GPIHBP1-LPL interaction, the consequences of mutations in the complex leading to chylomicronemia, and cutting-edge advancements in chylomicronemia treatment.
The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's alpha/beta-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal alpha-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.
Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.
        
Title: Angiopoietin-like protein 8: a multifaceted protein instrumental in regulating triglyceride metabolism Wen Y, Chen YQ, Konrad RJ Ref: Curr Opin Lipidol, :, 2023 : PubMed
PURPOSE OF REVIEW: The angiopoietin-like (ANGPTL) proteins ANGPTL3 and ANGPTL4 are critical lipoprotein lipase (LPL) inhibitors. This review discusses the unique ability of the insulin-responsive protein ANGPTL8 to regulate triglyceride (TG) metabolism by forming ANGPTL3/8 and ANGPTL4/8 complexes that control tissue-specific LPL activities. RECENT FINDINGS: After feeding, ANGPTL4/8 acts locally in adipose tissue, has decreased LPL-inhibitory activity compared to ANGPTL4, and binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin, which cleaves ANGPTL4/8 and other LPL inhibitors. This enables LPL to be fully active postprandially to promote efficient fatty acid (FA) uptake and minimize ectopic fat deposition. In contrast, liver-derived ANGPTL3/8 acts in an endocrine manner, has markedly increased LPL-inhibitory activity compared to ANGPTL3, and potently inhibits LPL in oxidative tissues to direct TG toward adipose tissue for storage. Circulating ANGPTL3/8 levels are strongly correlated with serum TG, and the ANGPTL3/8 LPL-inhibitory epitope is blocked by the TG-lowering protein apolipoprotein A5 (ApoA5). SUMMARY: ANGPTL8 plays a crucial role in TG metabolism by forming ANGPTL3/8 and ANGPTL4/8 complexes that differentially modulate LPL activities in oxidative and adipose tissues respectively. Selective ANGPTL8 inhibition in the context of the ANGPTL3/8 complex has the potential to be a promising strategy for treating dyslipidemia.
Cholesterol metabolism in the brain plays a crucial role in normal physiological function, and its aberrations are associated with cognitive dysfunction. The present study aimed to determine which cholesterol-related genes play a vital role in cognitive dysfunction and to dissect its underlying molecular mechanisms using a systems genetics approach in the BXD mice family. We first systematically analyzed the association of expression of 280 hippocampal genes related to cholesterol metabolism with cognition-related traits and identified lipoprotein lipase (Lpl) as a critical regulator. This was further confirmed by phenome-wide association studies that indicate Lpl associated with hippocampus volume residuals and anxiety-related traits. By performing expression quantitative trait locus mapping, we demonstrate that Lpl is strongly cis-regulated in the BXD hippocampus. We also identified -3,300 genes significantly (p < 0.05) correlated with the Lpl expression. Those genes are mainly involved in the regulation of neuron-related traits through the MAPK signaling pathway, axon guidance, synaptic vesicle cycle, and NF-kappa B signaling pathway. Furthermore, a protein-protein interaction network analysis identified several direct interactors of Lpl, including Rab3a, Akt1, Igf1, Crp, and Lrp1, which indicates that Lpl involves in the regulation of cognitive dysfunction through Rab3a-mediated synaptic vesicle cycle and Akt1/Igf1/Crp/Lrp1-mediated MAPK signaling pathway. Our findings demonstrate the importance of the Lpl, among the cholesterol-related genes, in regulating cognitive dysfunction and highlighting the potential signaling pathways, which may serve as novel therapeutic targets for the treatment of cognitive dysfunction.
The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's alpha/beta-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including beta2, beta3-alpha3, and the lid. Using pulse-labeling hydrogen-deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on beta3-alpha3 and progress to beta5 and beta4-alpha4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's alpha/beta-hydrolase domain (T (m) of 34.8 degreesC). GPIHBP1 binding dramatically increases LPL stability (T (m) of 57.6 degreesC), while ANGPTL4 lowers the onset of LPL unfolding by -20 degreesC, both for LPL and LPLGPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 degreesC but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.
        
Title: Lipoprotein Lipase and Its Regulators: An Unfolding Story Wu SA, Kersten S, Qi L Ref: Trends Endocrinol Metab, 32:48, 2021 : PubMed
Lipoprotein lipase (LPL) is one of the most important factors in systemic lipid partitioning and metabolism. It mediates intravascular hydrolysis of triglycerides packed in lipoproteins such as chylomicrons and very-low-density lipoprotein (VLDL). Since its initial discovery in the 1940s, its biology and pathophysiological significance have been well characterized. Nonetheless, several studies in the past decade, with recent delineation of LPL crystal structure and the discovery of several new regulators such as angiopoietin-like proteins (ANGPTLs), glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), lipase maturation factor 1 (LMF1) and Sel-1 suppressor of Lin-12-like 1 (SEL1L), have completely transformed our understanding of LPL biology.
Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-A resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.
Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 A resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.
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.
GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) within the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1-bound LPL is essential for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries, allowing the lipolytic processing of TRLs to proceed. In peripheral tissues, the intravascular processing of TRLs by the GPIHBP1-LPL complex is crucial for the generation of lipid nutrients for adjacent parenchymal cells. GPIHBP1 is absent from the capillaries of the brain, which uses glucose for fuel; however, GPIHBP1 is expressed in the capillaries of mouse and human gliomas. Importantly, the GPIHBP1 in glioma capillaries captures locally produced LPL. We use NanoSIMS imaging to show that TRLs marginate along glioma capillaries and that there is uptake of TRL-derived lipid nutrients by surrounding glioma cells. Thus, GPIHBP1 expression in gliomas facilitates TRL processing and provides a source of lipid nutrients for glioma cells.
Lipoprotein lipase (LPL), identified in the 1950s, has been studied intensively by biochemists, physiologists, and clinical investigators. These efforts uncovered a central role for LPL in plasma triglyceride metabolism and identified LPL mutations as a cause of hypertriglyceridemia. By the 1990s, with an outline for plasma triglyceride metabolism established, interest in triglyceride metabolism waned. In recent years, however, interest in plasma triglyceride metabolism has awakened, in part because of the discovery of new molecules governing triglyceride metabolism. One such protein-and the focus of this review-is GPIHBP1, a protein of capillary endothelial cells. GPIHBP1 is LPL's essential partner: it binds LPL and transports it to the capillary lumen; it is essential for lipoprotein margination along capillaries, allowing lipolysis to proceed; and it preserves LPL's structure and activity. Recently, GPIHBP1 was the key to solving the structure of LPL. These developments have transformed the models for intravascular triglyceride metabolism.
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.
In mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL-HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells.
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 is comprised of 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.
RATIONALE: GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia. OBJECTIVE: We sought to understand mechanisms by which GPIHBP1 mutations prevent LPL binding and lead to chylomicronemia. METHODS AND RESULTS: We expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1's Ly6 domain (including mutants identified in patients with chylomicronemia) led to the formation of disulfide-linked dimers and multimers. GPIHBP1 dimerization/multimerization was not unique to cysteine mutations; mutations in other amino acid residues, including several associated with chylomicronemia, also led to protein dimerization/multimerization. The loss of GPIHBP1 monomers is relevant to the pathogenesis of chylomicronemia because only GPIHBP1 monomers-and not dimers or multimers-are capable of binding LPL. One GPIHBP1 mutant, GPIHBP1-W109S, had distinctive properties. GPIHBP1-W109S lacked the ability to bind LPL but had a reduced propensity for forming dimers or multimers, suggesting that W109 might play a more direct role in binding LPL. In support of that idea, replacing W109 with any of 8 other amino acids abolished LPL binding-and often did so without promoting the formation of dimers and multimers. CONCLUSIONS: Many amino acid substitutions in GPIHBP1's Ly6 domain that abolish LPL binding lead to protein dimerization/multimerization. Dimerization/multimerization is relevant to disease pathogenesis, given that only GPIHBP1 monomers are capable of binding LPL.
Triglyceride-rich lipoproteins (TRLs) undergo lipolysis by lipoprotein lipase (LPL), an enzyme that is transported to the capillary lumen by an endothelial cell protein, GPIHBP1. For LPL-mediated lipolysis to occur, TRLs must bind to the lumen of capillaries. This process is often assumed to involve heparan sulfate proteoglycans (HSPGs), but we suspected that TRL margination might instead require GPIHBP1. Indeed, TRLs marginate along the heart capillaries of wild-type but not Gpihbp1(-)/(-) mice, as judged by fluorescence microscopy, quantitative assays with infrared-dye-labeled lipoproteins, and EM tomography. Both cell-culture and in vivo studies showed that TRL margination depends on LPL bound to GPIHBP1. Notably, the expression of LPL by endothelial cells in Gpihbp1(-)/(-) mice did not restore defective TRL margination, implying that the binding of LPL to HSPGs is ineffective in promoting TRL margination. Our studies show that GPIHBP1-bound LPL is the main determinant of TRL margination.
Brain lipid sensing is necessary to regulate energy balance. Lipoprotein lipase (LPL) may play a role in this process. We tested if hippocampal LPL regulated energy homeostasis in rodents by specifically attenuating LPL activity in the hippocampus of rats and mice, either by infusing a pharmacological inhibitor (tyloxapol), or using a genetic approach (adeno-associated virus expressing Cre-GFP injected into Lpl (lox/lox) mice). Decreased LPL activity by either method led to increased body weight gain due to decreased locomotor activity and energy expenditure, concomitant with increased parasympathetic tone (unchanged food intake). Decreased LPL activity in both models was associated with increased de novo ceramide synthesis and neurogenesis in the hippocampus, while intrahippocampal infusion of de novo ceramide synthesis inhibitor myriocin completely prevented body weight gain. We conclude that hippocampal lipid sensing might represent a core mechanism for energy homeostasis regulation through de novo ceramide synthesis.
        
Title: Angiopoietin-like protein 4 inhibition of lipoprotein lipase: evidence for reversible complex formation Lafferty MJ, Bradford KC, Erie DA, Neher SB Ref: Journal of Biological Chemistry, 288:28524, 2013 : PubMed
Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator.
Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.
GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, shuttles lipoprotein lipase (LPL) from subendothelial spaces to the capillary lumen. An absence of GPIHBP1 prevents the entry of LPL into capillaries, blocking LPL-mediated triglyceride hydrolysis and leading to markedly elevated triglyceride levels in the plasma (i.e., chylomicronemia). Earlier studies have established that chylomicronemia can be caused by LPL mutations that interfere with catalytic activity. We hypothesized that some cases of chylomicronemia might be caused by LPL mutations that interfere with LPL's ability to bind to GPIHBP1. Any such mutation would provide insights into LPL sequences required for GPIHBP1 binding. Here, we report that two LPL missense mutations initially identified in patients with chylomicronemia, C418Y and E421K, abolish LPL's ability to bind to GPIHBP1 without interfering with LPL catalytic activity or binding to heparin. Both mutations abolish LPL transport across endothelial cells by GPIHBP1. These findings suggest that sequences downstream from LPL's principal heparin-binding domain (amino acids 403-407) are important for GPIHBP1 binding. In support of this idea, a chicken LPL (cLPL)-specific monoclonal antibody, xCAL 1-11 (epitope, cLPL amino acids 416-435), blocks cLPL binding to GPIHBP1 but not to heparin. Also, changing cLPL residues 421 to 425, 426 to 430, and 431 to 435 to alanine blocks cLPL binding to GPIHBP1 without inhibiting catalytic activity. Together, these data define a mechanism by which LPL mutations could elicit disease and provide insights into LPL sequences required for binding to GPIHBP1.
The lipolytic processing of triglyceride-rich lipoproteins by lipoprotein lipase (LPL) is the central event in plasma lipid metabolism, providing lipids for storage in adipose tissue and fuel for vital organs such as the heart. LPL is synthesized and secreted by myocytes and adipocytes, but then finds its way into the lumen of capillaries, where it hydrolyzes lipoprotein triglycerides. The mechanism by which LPL reaches the lumen of capillaries has remained an unresolved problem of plasma lipid metabolism. Here, we show that GPIHBP1 is responsible for the transport of LPL into capillaries. In Gpihbp1-deficient mice, LPL is mislocalized to the interstitial spaces surrounding myocytes and adipocytes. Also, we show that GPIHBP1 is located at the basolateral surface of capillary endothelial cells and actively transports LPL across endothelial cells. Our experiments define the function of GPIHBP1 in triglyceride metabolism and provide a mechanism for the transport of LPL into capillaries.
The association of polymorphisms affecting lipid metabolism with the risk of myocardial infarction (MI) in type 2 diabetes mellitus was investigated. The Genetics, Outcomes and Lipids in type 2 Diabetes (GOLD) Study is a prospective, multicenter study, conducted on 990 patients presenting diabetes and MI (n=386), or diabetes without previous manifestation of stroke, peripheral or coronary arterial disease (n=604), recruited from 27 institutions in Brazil. APO A1 (A/G -75 and C/T +83) and APO C3 (C/G 3'UTR) non-coding sequences, CETP (Taq 1B), LPL (D9N), APO E (epsilon2, epsilon3, epsilon4,), PON-1 (Q192R), and two LCAT variants Arg(147)-->Trp and Tyr(171)-->Stop were tested by PCR-RFLP. There was a higher prevalence of LPL DN genotype (19% vs.12%, p=0.03) and a higher frequency of the N allele (11% vs. 7%) among subjects with MI when compared to controls, with an odds ratio of MI for carriers of 9N allele of 2.46 (95% CI=1.79-3.39, p<0.0001). This association was present in men and women, in non-smokers and in hypertensive patients. A logistic regression model including gender, duration of diabetes, systolic blood pressure, HDL-C, left ventricle hypertrophy and D9N polymorphism showed that the latter still remained significantly associated with MI (OR=1.50, 95% CI=1.02-2.25, p=0.049). These findings suggest that D9N polymorphism can be a useful risk marker for myocardial infarction and that further potential candidate genes should be screened for exploratory analysis and for future therapeutic intervention in diabetes.
High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.
        
Title: Lipoprotein lipase. Molecular model based on the pancreatic lipase x-ray structure: consequences for heparin binding and catalysis van Tilbeurgh H, Roussel A, Lalouel JM, Cambillau C Ref: Journal of Biological Chemistry, 269:4626, 1994 : PubMed
Lipoprotein lipase and pancreatic lipase have about 30% sequence identity, suggesting a similar tertiary fold. Three-dimensional models of lipoprotein lipase were constructed, based upon two recently determined x-ray crystal structures of pancreatic lipase, in which the active site was in an open and closed conformation, respectively. These models allow us to propose a few hypotheses on the structural determinants of lipoprotein lipase which are responsible for heparin binding, dimer formation, and phospholipase activity. The folding of the protein assembles a number of positive charge clusters at the back of the molecule, opposite the active site. These clusters probably form the heparin binding site, as confirmed by recent site-directed mutagenesis experiments. The active sites of lipoprotein lipase and pancreatic lipase look very similar, except for the lid (a surface loop covering the catalytic serine in the inactive state). A different open (active) conformation of the lid in both enzymes may be responsible for their differing substrate specificities. Predictions of the nature of the lipoprotein lipase dimer remain elusive, although our model enabled us to propose a few possibilities.
        
Title: Lipoprotein lipase enhances the interaction of low density lipoproteins with artery-derived extracellular matrix proteoglycans Edwards IJ, Goldberg IJ, Parks JS, Xu H, Wagner WD Ref: J Lipid Res, 34:1155, 1993 : PubMed
The association of plasma low density lipoproteins (LDL) with arterial proteoglycans (PG) is of key importance in LDL retention and modification in the artery wall. Lipoprotein lipase (LpL), the rate-limiting enzyme for hydrolysis of lipoprotein triglyceride, is known to bind both LDL and arterial PG. In the presence of LpL, cellular internalization and degradation of LDL is enhanced by a pathway initiated by interaction of LDL with a cell surface heparan sulfate proteoglycan. To determine whether LpL enhances the binding of LDL to arterial chondroitin sulfate (CS)PG and dermatan sulfate (DS)PG, the major extracellular PG of the artery wall, a microtiter plate assay was used to study LpL-PG-LDL interactions. Binding of LDL to both CSPG and DSPG was increased in the presence of LpL but differential effects were seen for the two PG. LpL enhanced the binding of LDL to CSPG a maximum of 20% and to DSPG a maximum of 40%. Heparin displacement of PG binding suggested a greater binding strength for DSPG-LpL-LDL with 0.25 micrograms heparin required to displace 50% of DSPG compared to 0.01 micrograms to displace 50% of CSPG. The greater enhancement of DSPG-LDL interaction by LpL is of particular interest since increases in DSPG correlate with the accumulation of aortic cholesterol. These data suggest that lipoprotein lipase may enhance the interaction of plasma low density lipoprotein with arterial chondroitin sulfate proteoglycan and dermatan sulfate proteoglycan and thus facilitate low density lipoprotein retention in the artery wall.
        
Title: Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix Eisenberg S, Sehayek E, Olivecrona T, Vlodavsky I Ref: J Clinical Investigation, 90:2013, 1992 : PubMed
Lipoprotein lipase enhances binding at 4 degrees C of human plasma lipoproteins (chylomicrons, VLDL, intermediate density lipoprotein, LDL, and HDL3) to cultured fibroblasts and hepG-2 cells and to extracellular matrix. Heparinase treatment of cells and matrix reduces the lipoprotein lipase enhanced binding by 90-95%. Lipoprotein lipase causes only a minimal effect on the binding of lipoproteins to heparan sulfate deficient mutant Chinese hamster ovary cells while it promotes binding to wild type cells that is abolished after heparinase treatment. With 125I-LDL, lipoprotein lipase also enhances uptake and proteolytic degradation at 37 degrees C by normal human skin fibroblasts but has no effect in heparinase-treated normal cells or in LDL receptor-negative fibroblasts. These observations prove that lipoprotein lipase causes, predominantly, binding of lipoproteins to heparan sulfate at cell surfaces and in extracellular matrix rather than to receptors. This interaction brings the lipoproteins into close proximity with cell surfaces and may promote metabolic events that occur at the cell surface, including facilitated transfer to cellular receptors.
Lipoprotein lipase (LPL) plays a crucial role in the regulation of lipoprotein metabolism by hydrolysing the core triglycerides of circulating chylomicrons and VLDL. Human, bovine, mouse, and guinea pig complementary DNA clones have recently been isolated and the organization of the human LPL gene is now known to comprise 10 exons spanning approximately 30 kb. Here we report a similar mutation on 21 alleles from 13 unrelated affected probands with LPL deficiency of French Canadian, English, Polish, German, Dutch, and East Indian ancestry. We show that an identical missense mutation within exon 5, resulting in an amino acid substitution of glutamic acid for glycine at position 188, is responsible for LPL deficiency in 21 of 88 LPL alleles assessed. This mutation alters an Ava II restriction site in exon 5 and will allow a rapid screening test for this mutation in patients with LPL deficiency. This mutation has occurred on the same haplotype in all the unrelated affected persons suggesting a common origin. The amino acid substitution lies within the longest segment of homology for LPL in different species and results in a protein that is catalytically defective.
Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.