Jennifer A. Beckstead

Structure and Interfacial Properties of Human Apolipoprotein A-V

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of α-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion.

Reconstituted high density lipoprotein enriched with the polyene antibiotic amphotericin B

The polyene antibiotic amphotericin B (AMB) is an effective antifungal agent whose therapeutic potential is limited by poor aqueous solubility and toxicity toward host tissues. Addition of apolipoprotein A-I to a multilamellar phospholipid vesicle dispersion containing 20% (w/w) AMB induces the formation of reconstituted high density lipoprotein (rHDL), with solubilization of the antibiotic. Density gradient ultracentrifugation resulted in flotation of the complexes to a density of 1.16 g/ml, and negative stain electron microscopy revealed a population of disk-shaped particles. Native gradient polyacrylamide gel electrophoresis indicated a particle diameter of ∼8.5 nm. Absorbance spectroscopy provided evidence for AMB integration into the lipid milieu. AMB-rHDLs were potent inhibitors of Saccharomyces cerevisiae growth, yielding 90% growth inhibition at <1 μg/ml yeast culture. In studies with pathogenic fungal species, similar growth inhibition characteristics were observed. Compared with AMB-deoxycholate micelles, AMB-rHDL displayed greatly attenuated red blood cell hemolytic activity and decreased toxicity toward cultured hepatoma cells. In in vivo studies in immunocompetent mice, AMB-rHDLs were nontoxic at 10 mg/kg, and they showed efficacy in a mouse model of candidiasis at concentrations as low as 0.25 mg/kg. These results indicate that AMB-rHDLs constitute a novel formulation that effectively solubilizes the antibiotic and elicits strong in vitro and in vivo antifungal activity with no observed toxicity at therapeutic doses.

Binding Preferences for GPIHBP1, a Glycosylphosphatidylinositol-Anchored Protein of Capillary Endothelial Cells

Objective—To define the ability of GPIHBP1 to bind other lipase family members and other apolipoproteins (apos) and lipoproteins. Methods and Results—GPIHBP1, a GPI-anchored lymphocyte antigen (Ly)6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1, but other members of the lipase family do not. We also examined the binding of apoAV-phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1s amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind high-density lipoprotein. Chylomicrons bind avidly to GPIHBP1-transfected Chinese hamster ovary cells, but this binding is dependent on GPIHBP1s ability to bind LPL within the cell culture medium. Conclusion—GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but does not bind apoAI or high-density lipoprotein. The ability of GPIHBP1-transfected Chinese hamster ovary cells to bind chylomicrons is mediated by LPL; chylomicron binding does not occur unless GPIHBP1 first captures LPL from the cell culture medium.

Binding Preferences for Glycosylphosphatidylinositol HBP1, a Glycosylphosphatidylinositol-Anchored Protein of Capillary Endothelial Cells

Objective—To define the ability of GPIHBP1 to bind other lipase family members and other apolipoproteins (apos) and lipoproteins. Methods and Results—GPIHBP1, a GPI-anchored lymphocyte antigen (Ly)6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1, but other members of the lipase family do not. We also examined the binding of apoAV-phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1s amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind high-density lipoprotein. Chylomicrons bind avidly to GPIHBP1-transfected Chinese hamster ovary cells, but this binding is dependent on GPIHBP1s ability to bind LPL within the cell culture medium. Conclusion—GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but does not bind apoAI or high-density lipoprotein. The ability of GPIHBP1-transfected Chinese hamster ovary cells to bind chylomicrons is mediated by LPL; chylomicron binding does not occur unless GPIHBP1 first captures LPL from the cell culture medium.

All trans retinoic acid nanodisks enhance retinoic acid receptor mediated apoptosis and cell cycle arrest in mantle cell lymphoma

Mantle cell lymphoma (MCL) is characterized by translocation t(11;14)(q13;q32), aggressive clinical behaviour, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel biological pathways. All trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand‐inducible transcription factors. The present study evaluated cell killing effects of ATRA‐enriched nanoscale delivery particles, termed nanodisks (ND), on MCL cell lines. Results show that ATRA‐ND induced cell death more effectively than naked ATRA (dimethyl sulphoxide) or empty ND. ATRA‐ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. The antioxidant, N‐acetylcysteine, inhibited ATRA‐ND induced apoptosis. Compared to naked ATRA, ATRA‐ND enhanced G1 growth arrest, up‐regulated p21and p27, and down regulated cyclin D1. At ATRA concentrations that induced apoptosis, expression levels of retinoic acid receptor‐α (RARα) and retinoid X receptor‐γ (RXRγ) were increased. Compared to naked ATRA, ATRA‐ND significantly stimulated transcriptional activity of RARA in a model carcinoma cell line. Furthermore, the RAR antagonist, Ro 41‐5253, inhibited ATRA‐ND induced ROS generation and prevented ATRA‐ND induced cell growth arrest and apoptosis. In summary, incorporation of ATRA into ND enhanced the biological activity of this retinoid in cell culture models of MCL.

The C Terminus of Apolipoprotein A-V Modulates Lipid-binding Activity *

Human apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To probe different regions of this 343-amino-acid protein, four single Trp apoA-V variants were prepared. The variant with a Trp at position 325, distal to the tetraproline sequence at residues 293–296, displayed an 11-nm blue shift in wavelength of maximum fluorescence emission upon lipid association. To evaluate the structural and functional role of this C-terminal segment, a truncated apoA-V comprising amino acids 1–292 was generated. Far UV circular dichroism spectra of full-length apoA-V and apoA-V-(1–292) were similar, with ∼50% α-helix content. In guanidine HCl denaturation experiments, both full-length and truncated apoA-V yielded biphasic profiles consistent with the presence of two structural domains. The denaturation profile of the lower stability component (but not the higher stability component) was affected by truncation. Truncated apoA-V displayed an attenuated ability to solubilize l-α-dimyristoylphosphatidylcholine phospholipid vesicles compared with full-length apoA-V, whereas a peptide corresponding to the deleted C-terminal segment displayed markedly enhanced kinetics. The data support the concept that the C-terminal region is not required for apoA-V to adopt a folded protein structure, yet functions to modulate apoA-V lipid-binding activity; therefore, this concept may be relevant to the mechanism whereby apoA-V influences plasma TG levels.

Intravenous Injection of Apolipoprotein A-V Reconstituted High-Density Lipoprotein Decreases Hypertriglyceridemia in apoav−/− Mice and Requires Glycosylphosphatidylinositol-Anchored High-Density Lipoprotein–Binding Protein 1

Objective—Apolipoprotein A-V (apoA-V), a minor protein associated with lipoproteins, has a major effect on triacylglycerol (TG) metabolism. We investigated whether apoA-V complexed with phospholipid in the form of a reconstituted high-density lipoprotein (rHDL) has potential utility as a therapeutic agent for treatment of hypertriglyceridemia (HTG) when delivered intravenously. Methods and Results—Intravenous injection studies were performed in genetically engineered mouse models of severe HTG, including apoav−/− and gpihbp1−/− mice. Administration of apoA-V rHDL to hypertriglyceridemic apoav−/− mice resulted in a 60% reduction in plasma TG concentration after 4 hours. This decline can be attributed to enhanced catabolism/clearance of very-low-density lipoprotein (VLDL), where VLDL TG and cholesterol were reduced ≈60%. ApoA-V that associated with VLDL after injection was also rapidly cleared. Site-specific mutations in the heparin-binding region of apoA-V (amino acids 186 to 227) attenuated apoA-V rHDL TG-lowering activity by 50%, suggesting that this sequence element is required for optimal TG-lowering activity in vivo. Unlike apoav−/− mice, injection of apoA-V rHDL into gpihbp1−/− mice had no effect on plasma TG levels, and apoA-V remained associated with plasma VLDL. Conclusion—Intravenously injected apoA-V rHDL significantly lowers plasma TG in an apoA-V deficient mouse model. Its intravenous administration may have therapeutic benefit in human subjects with severe HTG, especially in cases involving apoA-V variants associated with HTG.

Targeting nanodisks via a single chain variable antibody -apolipoprotein chimera

Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that alpha-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

Tweaking the cholesterol efflux capacity of reconstituted HDL

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.

The N-Terminus of Apolipoprotein A-V Adopts a Helix Bundle Molecular Architecture†

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic alpha-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.