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For three decades, the LPL–specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1 Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia–even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1 Enh/− mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), an endothelial cell protein, binds LPL in the subendothelial spaces and transports it to the capillary lumen. In Gpihbp1−/− mice, LPL remains stranded in the subendothelial spaces, causing hypertriglyceridemia, but how Gpihbp1−/− mice respond to metabolic stress (e.g., cold exposure) has never been studied. In wild-type mice, cold exposure increases LPL-mediated processing of triglyceride-rich lipoproteins (TRLs) in brown adipose tissue (BAT), providing fuel for thermogenesis and leading to lower plasma triglyceride levels. We suspected that defective TRL processing in Gpihbp1−/− mice might impair thermogenesis and blunt the fall in plasma triglyceride levels. Indeed, Gpihbp1−/− mice exhibited cold intolerance, but the effects on plasma triglyceride levels were paradoxical. Rather than falling, the plasma triglyceride levels increased sharply (from ∼4,000 to ∼15,000 mg/dl), likely because fatty acid release by peripheral tissues drives hepatic production of TRLs that cannot be processed. We predicted that the sharp increase in plasma triglyceride levels would not occur in Gpihbp1−/−Angptl4−/− mice, where LPL activity is higher and baseline plasma triglyceride levels are lower. Indeed, the plasma triglyceride levels in Gpihbp1−/−Angptl4−/− mice fell during cold exposure. Metabolic studies revealed increased levels of TRL processing in the BAT of Gpihbp1−/−Angptl4−/− mice.

ApoC-III is often assumed to retard the intravascular processing of triglyceride-rich lipoproteins (TRLs) by inhibiting LPL, but that view is based largely on studies of free LPL. We now recognize that intravascular LPL is neither free nor loosely bound, but instead is tightly bound to glycosylphosphatidylinositol-anchored HDL-binding protein 1 (GPIHBP1) on endothelial cells. Here, we revisited the effects of apoC-III on LPL, focusing on apoC-IIIʼns capacity to affect the activity of GPIHBP1-bound LPL. We found that TRLs from APOC3 transgenic mice bound normally to GPIHBP1-bound LPL on cultured cells in vitro and to heart capillaries in vivo. However, the triglycerides in apoC-III–enriched TRLs were hydrolyzed more slowly by free LPL, and the inhibitory effect of apoC-III on triglyceride lipolysis was exaggerated when LPL was bound to GPIHBP1 on the surface of agarose beads. Also, recombinant apoC-III reduced triglyceride hydrolysis by free LPL only modestly, but the inhibitory effect was greater when the LPL was bound to GPIHBP1. A mutant apoC-III associated with low plasma triglyceride levels (p.A23T) displayed a reduced capacity to inhibit free and GPIHBP1-bound LPL. Our results show that apoC-III potently inhibits triglyceride hydrolysis when LPL is bound to GPIHBP1.

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease—chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease

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.

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1–297) and a carboxyl-terminal domain (residues 298–448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues ∼403–438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues ∼403–438) were not sufficient for 88B8 binding; upstream sequences (residues 298–400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.

Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA) in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB). To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model.

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.