The triglycerides in chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of the capillaries. However, the endothelial cell molecule that facilitates chylomicron processing by LpL has not yet been defined. Here, we show that glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) plays a critical role in the lipolytic processing of chylomicrons. Gpihbp1-deficient mice exhibit a striking accumulation of chylomicrons in the plasma, even on a low-fat diet, resulting in milky plasma and plasma triglyceride levels as high as 5000 mg/dl. Normally, Gpihbp1 is expressed highly in heart and adipose tissue, the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. These studies strongly suggest that GPIHBP1 is an important platform for the LpL-mediated processing of chylomicrons in capillaries.

SUMMARY 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 represents one of the most persistent mysteries 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 CRISPR-Cas9 system provides unprecedented genome editing capabilities. However, off-target effects lead to sub-optimal usage and additionally are a bottleneck in the development of therapeutic uses. Herein, we introduce the first machine learning-based approach to off-target prediction, yielding a state-of-the-art model for CRISPR-Cas9 that outperforms all other guide design services. Our approach, Elevation, consists of two interdependent machine learning models—one for scoring individual guide-target pairs, and another which aggregates these guide-target scores into a single, overall summary guide score. Through systematic investigation, we demonstrate that Elevation performs substantially better than competing approaches on both tasks. Additionally, we are the first to systematically evaluate approaches on the guide summary score problem; we show that the most widely-used method performs no better than random at times, whereas Elevation consistently outperformed it, sometimes by an order of magnitude. We also introduce an evaluation method that balances errors between active and inactive guides, thereby encapsulating a range of practical use cases; Elevation is consistently superior to other methods across the entire range. Finally, because of the large scale and computational demands of off-target prediction, we have developed a cloud-based service for quick retrieval. This service provides end-to-end guide design by also incorporating our previously reported on-target model, Azimuth. (https://crispr.ml:please treat this web site as confidential until publication).

Objective-GPIHBP1 is an endothelial cell protein that binds lipoprotein lipase (LPL) and chylomicrons. Because GPIHBP1 deficiency causes chylomicronemia in mice, we sought to determine whether some cases of chylomicronemia in humans could be attributable to defective GPIHBP1 proteins. Methods and Results-Patients with severe hypertriglyceridemia (nϭ60, with plasma triglycerides above the 95th percentile for age and gender) were screened for mutations in GPIHBP1. A homozygous GPIHBP1 mutation (c.344AϾC) that changed a highly conserved glutamine at residue 115 to a proline (p.Q115P) was identified in a 33-year-old male with lifelong chylomicronemia. The patient had failure-to-thrive as a child but had no history of pancreatitis. He had no mutations in LPL, APOA5, or APOC2. The Q115P substitution did not affect the ability of GPIHBP1 to reach the cell surface. However, unlike wild-type GPIHBP1, GPIHBP1-Q115P lacked the ability to bind LPL or chylomicrons (d Ͻ 1.006 g/mL lipoproteins from Gpihbp1 Ϫ/Ϫ mice). Mouse GPIHBP1 with the corresponding mutation (Q114P) also could not bind LPL. Conclusions-A homozygous missense mutation in GPIHBP1 (Q115P) was identified in a patient with chylomicronemia.The mutation eliminated the ability of GPIHBP1 to bind LPL and chylomicrons, strongly suggesting that it caused the patient's chylomicronemia. See accompanying article on page 792Mice lacking GPIHBP1 manifest severe chylomicronemia, even on a low-fat chow diet, with plasma triglycerides Ͼ2000 mg/dL. 2 GPIHBP1 is found on the luminal surface of capillaries in heart, skeletal muscle, and adipose tissue, 2 where the lipolytic processing of triglyceride-rich lipoproteins occurs. 3 Transfection of a GPIHBP1 expression vector into CHO cells confers the ability to bind LPL, chylomicrons, as well as apo-AV-phospholipid disks. 2 The ability of GPIHBP1-expressing cells to bind LPL and chylomicrons suggested that GPIHBP1 might function as a platform for lipolysis on endothelial cells. 2 Two structural features of GPIHBP1 are important in the binding of LPL and chylomicrons. The first is an aminoterminal acidic domain, approximately 25 amino acids in length. Mutant GPIHBP1 proteins lacking all or part of the acidic domain are unable to bind LPL and chylomicrons. 4 The second is a Lymphocyte antigen 6 (Ly6) domain. Ly6 motifs, which contain either 8 or 10 cysteines with a characteristic spacing pattern, are found in a number of GPI-anchored proteins, for example CD59 and the urokinase-type plasminogen activator receptor (UPAR). 5 When the Ly6 domain of GPIHBP1 is replaced with the Ly6 domain from CD59, the chimeric protein reaches the cell surface but cannot bind LPL, even though the acidic domain of GPIHBP1 is intact. 4 All mammalian GPIHBP1 proteins share the acidic domain and the Ly6 domain (with 10 conserved cysteines). 6 The highest level of amino acid conservation lies within a portion of the Ly6 domain (residues 101 to 121 in human GPIHBP1, which contains the 6th and 7th cysteines of the Ly6 motif). 6 The finding of chylomicronemia...