Family Report for: Lipoprotein_Lipase

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.

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 hydrogendeuterium 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.

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.

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.

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 hydrogendeuterium 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.

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.

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.

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.

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.

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.