The conformation of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. 13C NMR studies of lysine ionization behavior.

To elucidate the molecular details of how high density lipoprotein (HDL) microstructure affects the conformation of apolipoprotein (apo) A-I in various classes of HDL particles, apoA-I structure in homogeneous recombinant HDL (rHDL) complexes containing palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate has been investigated by NMR spectroscopy of [13C]lysine-labeled apoA-I. All Lys residues in rHDL apoA-I were labeled with 13C by reductive methylation, and then their ionization behavior was characterized by 13C NMR spectroscopy. Four discoidal particles were prepared to contain from 64 to 256 molecules of POPC and 2 molecules of apoA-I; their major diameters ranged from 9.3 to 12.1 nm. (13CH3)2-Lys resonances from apoA-I in discoidal complexes exhibit six distinct chemical shifts at pH 10. The various Lys have pKa values ranging from 8.3 to 10.5, indicating that they exist in different microenvironments. More than 80% of the Lys residues in small (9.3 nm) discoidal particles titrate at a significantly lower pH than in the large (12.1 nm) discoidal particles. This indicates that apoA-I has a different conformation on the differently size discs. Two spherical particles were prepared with POPC:cholesteryl oleate:apoA-I molar stoichiometries of 56:16:2 and 232:84:4 and diameters of 7.4 and 12.6 nm, respectively. On spherical rHDL, apoA-I (13CH3)2-Lys resonances exhibit five distinct chemical shifts at pH 10. The titration behavior of apoA-I Lys residues is the same in small and large spherical particles, indicating that apoA-I conformation is similar on the two particles. The Lys microenvironments indicate that the conformation of apoA-I in discoidal complexes is dependent on particle size and that these conformations are substantially different from that of apoA-I on spherical complexes. Lys microenvironments in discoidal complexes differ from that of spherical complexes by 4 to 5 ysines which titrate with relatively low pKa values on discs. This reflects apparent differences in conformation in the NH2-terminal one-third of apoA-I on discs and spheres.     

Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer

Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.     

Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.

The details of how high density lipoprotein (HDL) microstructure affects the conformation and net charge of apolipoprotein (apo) A-I in various classes of HDL particles have been investigated in homogeneous recombinant HDL (rHDL) particles containing apoA-I, palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate. Isothermal denaturation with guanidine HCl was used to monitor alpha-helix structural stability, whereas electrokinetic analyses and circular dichroism were used to determine particle charge and apoA-I secondary structure, respectively. Electrokinetic analyses show that at pH 8.6 apoA-I has a net negative charge on discoidal (POPC.apoA-I) particles (-5.2 electronic units/mol of apoA-I) which is significantly greater than that of apoA-I either free in solution or on spherical (POPC.cholesteryl oleate.apoA-I) rHDL (approximately -3.5 electronic units). Raising the POPC content (32-128 mol/ml of apoA-I) of discoidal particles 1) increases the particle major diameter from 9.3 to 12.1 nm, 2) increases the alpha-helix content from 62 to 77%, and 3) stabilizes the helical segments by increasing the free energy of unfolding (delta GD degree) from 1.4 to 3.0 kcal/mol of apoA-I. Raising the POPC content (28-58 mol/mol of apoA-I) of spherical particles 1) increases the particle diameter from 7.4 to 12.6 nm, 2) increases the percent alpha-helix from 62 to 69%, and 3) has no significant effect on delta GD degree (2.2 kcal/mol of apoA-I). This study shows that different HDL subspecies maintain particular apoA-I conformations that confer unique charge and structural characteristics on the particles. It is likely that the charge and conformation of apoA-I are critical molecular properties that modulate the metabolism of HDL particles and influence their role in cholesterol transport.     

Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin: cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells

Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3μg/mL vs. 0.59±0.06μg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug.     

Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high-density lipoprotein phospholipids

Hepatic lipase (HL) and endothelial lipase (EL) are both members of the triglyceride lipase gene family. HL hydrolyzes phospholipids and triglycerides in triglyceride-rich lipoproteins and high-density lipoproteins (HDL). EL hydrolyzes HDL phospholipids and has low triglyceride lipase activity. The aim of this study was to determine if HL and EL hydrolyze different HDL phospholipids and whether HDL phospholipid composition regulates the interaction of EL and HL with the particle surface. Spherical, reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoylphosphatidylcholine (PDPC) as the only phospholipid, apolipoprotein A-I as the only apolipoprotein, and either cholesteryl esters (CE) only or mixtures of CE and triolein (TO) in their core were prepared. The rHDL were similar in size and had comparable core lipid/apoA-I molar ratios. The CE-containing rHDL were used to determine the kinetics of HL- and EL-mediated phospholipid hydrolysis. For HL the V(max) of phospholipid hydrolysis for (POPC)rHDL > (PLPC)rHDL approximately (PDPC)rHDL > (PAPC)rHDL, while the K(m)(app) for (POPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (PAPC)rHDL. For EL the V(max) for (PDPC)rHDL > (PAPC)rHDL > (PLPC)rHDL approximately (POPC)rHDL, while the K(m)(app) for (PAPC)rHDL approximately (PLPC)rHDL > (POPC)rHDL > (PDPC)rHDL. The kinetics of EL- and HL-mediated TO hydrolysis was determined using rHDL that contained TO in their core. For HL the V(max) of TO hydrolysis for (PLPC)rHDL > (POPC)rHDL > (PAPC)rHDL > (PDPC)rHDL, while the K(m)(app) for (PLPC)rHDL > (POPC)rHDL approximately (PAPC)rHDL > (PDPC)rHDL. For EL the V(max) and K(m)(app) for (PAPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (POPC)rHDL. These results establish that EL and HL have different substrate specificities for rHDL phospholipids and that their interactions with the rHDL surface are regulated by phospholipids.     

Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe

Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound states. Spectroscopic approaches, including circular dichroism and various fluorescence methods, were used to examine the properties of the mutant proteins and of their covalent adducts with the fluorescence probe. The mutations themselves had no effect on the structure and stability of apoA-I in the lipid-free state and in reconstituted HDL (rHDL) complexes. Furthermore, covalent modification with acrylodan did not alter the properties of the apoA-I variants in the lipid-bound state nor in the lipid-free A232C mutant, but it affected the structure and local stability of the lipid-free protein in the D9C mutant. Fluorescence results using the acrylodan probe confirmed a well-organized structure in the N-terminal region of apoA-I. Also, they suggested a three-dimensional structure in the C-terminal region, stabilized by protein-protein contacts. When Trp residues and acrylodan were used as donor-acceptor pairs for fluorescence resonance energy transfer (FRET), average distances could be measured. Both intensity and lifetime changes of the Trp emission indicated a protein folding in solution that brings the C-terminus of the protein near the Trp residues in the N-terminal half of the sequence. Also, the N- and C-terminal domains of apoA-I appeared to be near each other in rHDL having two apoA-I per particle.     

Influence of phospholipid depletion on the size, structure, and remodeling of reconstituted high density lipoproteins

This study shows that phospholipid depletion has a major impact on the size and structure of spherical, reconstituted high density lipoproteins (rHDL) and their remodeling by cholesteryl ester transfer protein (CETP). Spherical rHDL, 9.2 nm in diameter with a phospholipid/cholesteryl ester/unesterified cholesterol/apolipoprotein A-I (apoA-I) (PL/CE/UC/A-I) molar ratio of 37.3/24.5/4.1/1.0, were depleted progressively of phospholipids by incubation with phospholipase A(2). After 30 min of incubation the PL/CE/UC/A-I molar ratio of the rHDL was 8.0/31.2/4.4/1.0 and their diameter had decreased to 8.0 nm. Comparable changes in rHDL size and composition were also apparent when the incubations were carried out in the presence of other lipoprotein classes and lipoprotein-deficient plasma. The changes in size and composition were not accompanied by the dissociation of apoA-I from the rHDL. Phospholipid depletion did not affect rHDL surface charge or the structure and stability of apoA-I. The remodeling of unmodified and phospholipid-depleted rHDL by CETP was also investigated. When the rHDL were incubated for 3 h with CETP and Intralipid, transfers of core lipids between the phospholipid-depleted rHDL and Intralipid were decreased relative to unmodified rHDL. This difference was no longer apparent when the incubations were extended beyond 3 h. In these incubations apoA-I dissociated from the phospholipid-depleted and unmodified rHDL at 3 and 12 h, respectively. At 24 h the respective diameters of the unmodified rHDL and phospholipid-depleted rHDL were 8.0 and 7.8 nm. In conclusion, phospholipid depletion has a major impact on rHDL size and their remodeling by CETP.     

ApoA-I structure on discs and spheres. Variable helix registry and conformational states

Apolipoprotein A-I (apoA-I) readily forms discoidal high density lipoprotein (HDL) particles with phospholipids serving as an ideal transporter of plasma cholesterol. In the lipid-bound conformation, apoA-I activates the enzyme lecithin:cholesterol acyltransferase stimulating the formation of cholesterol esters from free cholesterol. As esterification proceeds cholesterol esters accumulate within the hydrophobic core of the discoidal phospholipid bilayer transforming it into a spherical HDL particle. To investigate the change in apoA-I conformation as it adapts to a spherical surface, fluorescence resonance energy transfer studies were performed. Discoidal rHDL particles containing two lipid-bound apoA-I molecules were prepared with acceptor and donor fluorescent probes attached to cysteine residues located at specific positions. Fluorescence quenching was measured for probe combinations located within repeats 5 and 5 (residue 132), repeats 5 and 6 (residues 132 and 154), and repeats 6 and 6 (residue 154). Results from these experiments indicated that each of the 2 molecules of discoidal bound apoA-I exists in multiple conformations and support the concept of a "variable registry" rather than a "fixed helix-helix registry." Additionally, discoidal rHDL were transformed in vitro to core-containing particles by incubation with lecithin:cholesterol acyltransferase. Compositional analysis showed that core-containing particles contained 11% less phospholipid and 633% more cholesterol ester and a total of 3 apoA-I molecules per particle. Spherical particles showed a lowering of acceptor to donor probe quenching when compared with starting rHDL. Therefore, we conclude that as lipid-bound apoA-I adjusts from a discoidal to a spherical surface its intermolecular interactions are significantly reduced presumably to cover the increased surface area of the particle.     

Structural and functional properties of apolipoprotein A-I mutants containing disulfide-linked cysteines at positions 124 or 232

Recombinant Cys mutants of apolipoprotein A-I (apoA-I) (A124C and A232C) have been prepared in disulfide-linked forms in order to assess the effects of unnatural covalent constraints on the folding of apoA-I in solution, its ability to bind lipids, form HDL-like particles, activate LCAT, and undergo structural adaptations to changing lipid contents. Both mutants, in dimer form, were shown to fold similarly to plasma apoA-I in solution, but had a slightly decreased alpha-helix content and no evidence of intermonomer interactions. All forms of the mutants bound to and disrupted dimyristoylphosphatidylcholine (DMPC) liposomes with similar kinetics and efficiency to plasma apoA-I, and formed reconstituted HDL (rHDL) particles with palmitoyloleoylphosphatidylcholine (POPC) in high yields at three different ratios of lipid/protein. While the monomeric mutants produced identical rHDL to plasma apoA-I, the disulfide-linked dimers had distinct particle distributions from each other and from native apoA-I. The A124C-dimer formed rHDL with diameters of 86 and 78 A, while the A232C-dimer predominantly formed 96 A rHDL. These particles, and particles containing plasma apoA-I (96 and 78 A), were purified prior to structural and functional analyses. The structural properties of particles with similar diameters were comparable, as were their reactivities with LCAT; however, their ability to undergo structural rearrangements differed. The larger rHDL particles (96 and 86 A) containing native apoA-I or A124C-dimer, rearranged into smaller 78 A particles, while the 96 A particles containing A232C-dimer were resistant to rearrangement and did not form 78 A particles. From the results, it is concluded that synthetic, random disulfide-linked dimers of apoA-I have many properties analogous to those of the naturally occurring Cys mutants, apoA-I-Milano and apoA-I-Paris, which are thought to have antiatherogenic effects in vivo. Also, the results have implications for current models of rHDL structure.     

A key point mutation (V156E) affects the structure and functions of human apolipoprotein A-I

A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more alpha-helical and formed a larger complex (110 A) than control apoA-I (97 A). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.     

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.

Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling. Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT.     

The structure of apolipoprotein A-II in discoidal high density lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.     

Cell cholesterol efflux to reconstituted high-density lipoproteins containing the apolipoprotein A-IMilano dimer

The apolipoprotein A-IMilano (apoA-IM) is a molecular variant of apoA-I characterized by the Arg(173)-->Cys substitution, resulting in the formation of homodimers A-IM/A-IM. The introduction of the interchain disulfide bridge in the A-IM dimer limits the apolipoprotein conformational flexibility and restricts HDL particle size heterogeneity, thus possibly affecting HDL function in lipid metabolism and atherosclerosis protection. To investigate whether the structural changes in A-IM/A-IM affect apoA-I capacity for cell cholesterol uptake, we tested the ability of four reconstituted HDL (rHDL), that contained either apoA-I or A-IM/A-IM, to remove cholesterol from Fu5AH hepatoma cells and cholesterol-loaded murine primary macrophages (MPM). As the HDL particle size is known to affect the rHDL capacity for cell cholesterol uptake, the reconstitution conditions were carefully selected to produce two sets of rHDL particles of small and large size (7.8 and 12.5 nm in diameter). The small A-IM/A-IM rHDL were more efficient than the corresponding apoA-I particles as acceptors of membrane cholesterol from Fu5AH cells and MPM, and as inhibitors of cholesterol esterification in MPM. The large rHDL and the lipid-free apolipoproteins displayed instead similar capacities for cell cholesterol efflux. These results suggest that cell cholesterol efflux to rHDL particles of different size occurs through different mechanisms. Large HDL accommodate and retain the cholesterol molecules that have desorbed from the cell membrane into the extracellular fluid, in a process that is less sensitive to protein conformation. Small HDL accelerate the desorption of cholesterol from the cell membrane, in a process that is influenced by the conformation of the proteins on the surface of the acceptor particle. The enhanced efficiency of small A-IM/A-IM rHDL seems related to the peculiar structure of the protein on the rHDL surface, with a hydrophobic C-terminal domain extending out of the rHDL particle, available for anchoring the acceptor to the plasma membrane.     

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I

Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and beta-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 A, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed.     

Evidence for a central apolipoprotein A-I domain loosely bound to lipids in discoidal lipoproteins that is capable of penetrating the bilayer of phospholipid vesicles

Previous evidence indicated that discoidal reconstituted high density lipoproteins (rHDL) of apolipoprotein A-I (apoA-I) can interact with lipid membranes (Tricerri, M. A., Córsico, B., Toledo, J. D., Garda, H. A., and Brenner, R. R. (1998) Biochim. Biophys. Acta 1391, 67-78). With the aim of studying this interaction, photoactivable reagents and protein cleavage with CNBr and hydroxylamine were used. The generic hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine gave information on the apoA-I regions in contact with the lipid phase in the rHDL discs. Two protein regions loosely bound to lipids were detected: a C-terminal domain and a central one located between residues 87 and 112. They consist of class Y amphipathic alpha-helices that have a different distribution of the charged residues in their polar faces by comparison with class A helices, which predominate in the rest of the apoA-I molecule. The phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoro-methyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, which does not undergo significant exchange between membranes and lipoproteins, was used to identify the apoA-I domain directly involved in the interaction of rHDL discs with membranes. By incubating either rHDL or lipid-free apoA-I with lipid vesicles containing 125I-TID-PC, only the 87-112 apoA-I segment becomes labeled after photoactivation. These results indicate that the central domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes.     

Evidence that apolipoprotein A-I facilitates hepatic lipase-mediated phospholipid hydrolysis in reconstituted HDL containing apolipoprotein A-II

This study examines hepatic lipase (HL) mediated phospholipid hydrolysis in mixtures of apolipoprotein-specific, spherical reconstituted high-density lipoproteins (rHDL). We have shown previously that apolipoprotein A-I (apoA-I) and apoA-II have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in well-characterized, homogeneous preparations of spherical rHDL [Hime, N. J., Barter, P. J., and Rye, K.-A. (1998) J. Biol. Chem. 273, 27191-27198]. In the present study, phospholipid hydrolysis was assessed in mixtures of rHDL containing either apoA-I only, (A-I)rHDL, apoA-II only, (A-II)rHDL, or both apoA-I and apoA-II, (A-I/A-II)rHDL. The rHDL contained trace amounts of radiolabeled phospholipid, and hydrolysis was measured as the formation of radiolabeled nonesterified fatty acids (NEFA). As predicted from our previous kinetic studies, the (A-II)rHDL acted as competitive inhibitors of HL-mediated phospholipid hydrolysis in (A-I)rHDL. Less expected was the observation that the rate of phospholipid hydrolysis in (A-II)rHDL was enhanced when (A-I)rHDL were also present in the incubation mixture. The rate of phospholipid hydrolysis in (A-I/A-II)rHDL was also greater than in (A-II)rHDL, indicating that apoA-I enhances phospholipid hydrolysis when it is present as a component of (A-I/A-II)rHDL. It is concluded that apoA-I enhances HL-mediated phospholipid hydrolysis in apoA-II containing rHDL, irrespective of whether the apoA-I is present in the same particle as the apoA-II [as in (A-I/A-II)rHDL] or whether it is present as a component of a different particle, such as when (A-I)rHDL are added to incubations of (A-II)rHDL.     

Different Functional and Structural Characteristics between ApoA-I and ApoA-4 in Lipid-Free and Reconstituted HDL State: ApoA-4 Showed Less Anti-Atherogenic Activity

Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS₃-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties.     

Apolipoprotein A-I helix 6 negatively charged residues attenuate lecithin-cholesterol acyltransferase (LCAT) reactivity

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.     

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.