Lipid-bound apoA-I Research Articles

Significance of Lipid-Free and Lipid-Associated ApoA-I in Cellular Cho-lesterol Efflux.

The structure and stability of apolipoprotein (apo)A-I, the major apolipoprotein of human plasma high-density lipoproteins (HDL), determine the efficiency of the protein in the process of HDL generation and affect HDL properties in binding and exchanging its constituents, thus playing an essential role in reverse cholesterol transport. The equilibrium stability of an apoA-I molecule at the lipid interface (12.7 kcal/mol) predicted by a thermodynamic cycle for apolipoprotein folding-unfolding in water and at interface, largely exceeds apoA-I helix stability in HDL against chemical denaturation (3-5 kcal/mol). An ensemble of structures of lipid-bound apoA-I with different stabilities is assumed to exist. The conformational transitions between apoA-I conformers in water and lipid phases correspond to Lumry-Eyring model OL ⇔ CL ⇒ MW, where OL and CL are open and closed structures of HDLbound apoA-I, and MW is the molten globule in water. The model includes the reversible foldingunfolding transitions of N- and C-domains at HDL interface and apolipoprotein irreversible dissociation. We gathered published data on cholesterol efflux for apoA-I proteins with missense mutations in C-domain and calculated the stability of these mutants as a change of free energy relative to a wild type protein. Significant negative correlation was found between this stability and the efficiency of cAMP-stimulated cholesterol efflux. Thus, besides the known role of C-domain hydrophobicity, structure-destabilizing changes may significantly contribute to ABCA1-mediated cholesterol efflux by free apolipoprotein.

Abstract 27: Structure of Lipid-free and Lipid-bound Apolipoprotein A-I Determined by Stable Isotope-assisted Cross-linking and Small Angle X-ray Scattering

Apolipoprotein (apo)A-I, the major protein constituent of high density lipoproteins (HDL), acts as a conformationally dynamic scaffold on the surface of the particle. Evidence suggests apoA-I conformation dictates HDL functionality and capacity to interface with other HDL proteins; however, the details of apoA-I structure and HDL formation are not completely understood. X-ray crystal structures of truncated forms of apoA-I and its cousin, apoA-IV, show reciprocal domain swapping structures in the lipid-free state. Recently, we showed the truncated mutant of apoA-I is also highly dynamic in solution with a flexible N-terminus. The purpose of this study was to a) derive a structural model of wild-type (WT) apoA-I using the new in-solution truncation model as a template and b) determine the relationship between monomeric, and oligomeric forms of lipid-free WT apoA-I. Using a novel isotope labeling strategy and chemical cross-linking, we defined spatial relationships within and between different molecules of WT apoA-I. The cross-linking patterns revealed a high degree of structural similarity between WT apoA-I oligomers. We found that monomeric and dimeric WT apoA-I structures were similar, but not identical, to those predicted by the crystal and solution structures of truncated apoA-I. Using cross-linking data, we derived two interconverting structures each for monomeric WT apoA-I, both containing dynamic N- and C-terminal regions. Similar structures were generated for WT dimeric and trimeric species. Small angle X-ray scattering was used to derive molecular envelopes of the molecules and refine the arrangements to propose the most detailed molecular models of lipid-free WT apoA-I to date. The models were used in conjunction with the reported double-belt and trefoil structures reconstituted HDL to reveal evidence of a parsimonious relationship between lipid-free and lipid-bound WT apoA-I. We propose a structural framework of WT apoA-I self-association and lipidation, centric to the C-terminal region of the molecule, that forms a basis for understanding HDL particle formation at the cell surface.

Abstract 223: The Structural Organization of Apolipoprotein A-I in Reconstituted High-Density Lipoprotein Discs Using Stable Isotope-Assisted Cross-Linking

X-ray crystallography studies of lipid-free apolipoprotein A-I (apoA-I) and related apoA-IV have shown that the proteins form a reciprocating dimer which could be directly related to the structural organization of lipid-bound apoA-I. Previous work has demonstrated the success of chemical cross-linking combined with mass spectrometry to derive distance constraints for molecular modeling of apoA-I in reconstituted high density lipoprotein (HDL) particles. However, these studies have been limited by i) the relative paucity of distance constraint data, and ii) ambiguities in determining the molecular span of the cross-links. Here, we have addressed the first issue by using an affinity-tagged cross-linker to purify cross-linked peptides away from non-modified peptides prior to sampling with mass spectrometry. This essentially boosts the signal from cross-linked peptides of interest that would have otherwise been suppressed by detection of non-modified peptides. Using this method, we identified >25 cross-linked peptides; the most reported to date for these particles. To address the issue of molecular span, we used a stable isotopic-labeling approach in which wild-type human recombinant apoA-I ( 14 N) was mixed 1:1 with an isotopically labeled ( 15 N) apoA-I. Analysis of the isotopic patterns of the resulting mass spectra allowed the unambiguous determination of the molecular span of the cross-linked peptides; i.e. whether the links were within the same molecule of apoA-I (intramolecular) or between the dimers of apoA-I (intermolecular). This information will be used to derive a detailed novel model of lipid-bound apoA-I in reconstituted HDL, an important step in our overall understanding of how native HDL species are organized and how they interact with plasma remodeling factors and cell surface proteins.

Mutation mapping of apolipoprotein A-I structure assisted with the putative cholesterol recognition regions

Twenty-nine from 52 missense mutations in apoA-I gene are predicted to be deleterious by both SIFT and PolyPhen-2 algorithms. Among those, eight mutations with a prominent change in structure stability as modeled by the SDM tool for both lipid-free (Mei and Atkinson (2011) PDB ID: 3R2P) and HDL-bound (Wu et al. (2009) PDB ID: 3K2S) apoA-I, are referred as structural. The remaining mutations with a preferential location in a long intrinsically disordered region, predicted by the SPINE-D and DNdisorder tools, may influence the functional sites. Among structural mutations, five amyloidosis-only-related mutations, significant in a lipid-free structure, are located in 1–90 region. Six amyloidosis- and hypoalphalipoproteinemia-associated mutations, differently significant in two chains of lipid-bound apoA-I, are distributed among the N-domain. Six cholesterol recognition putative motifs (5 CRAC/1 CCM) in apoA-I structure are suggested to interact with cholesterol. Among those, the K40-W50 partially conserved CCM sequence with a putative recognition feature, predicted by the MoRF tool, may underlie cholesterol binding to lipid-free apoA-I, the binding triggering the disorder-to-order transition within MoRF. Thus, the impairment of helix formation and accelerated protein aggregation may underlie the amyloidogenic effect of W50R substitution. Also, D102H substitution in conserved CRAC2 V97-K106 sequence may be harmful in reverse cholesterol transport. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in HDL-bound apoA-I. The influence of specific mutation on apoA-I structure and mutated apolipoprotein switch between different pathologies is suggested to depend on the surrounding phase properties.

Abstract 412: In Silico Structural and Metabolic Profiling of Non-Synonymous Single Nucleotide Polymorphisms in APOA-I Gene

Mature apoA-I contains 243 residues organized in two-domains as N-terminal four-helix bundle and C-terminal two helices. Cholesterol interaction with both lipid-free and lipid-bound apoA-I may be one of the key events in amyloidosis and reverse cholesterol transport (RCT). By the SIFT score, 20 non-synonymous missense mutations in APOA-I gene, located almost exclusively in N-domain, may impair apoA-I function. A majority (4 from 5) of putative cholesterol recognition amino acid consensus (CRAC: L/V-X(1-5)-Y-X(1-5)-R/K) and a single cholesterol consensus motif (CCM: R/K-X(2-6)-I/L/V-X(3)-W/Y) aligned within/between helices in the bundle. Importantly, CRAC2 in apoA-I co-aligned with CRAC2 in apoE and CRAC4 in apoA-IV. A short four amino acid sequence L44-L47 may present disorder-to-order transitioning binding region in apoA-I as predicted by molecular recognition feature (MoRF). Indeed, CCM corresponded to unstructured region with low stability in lipid-free apoA-I. Stabilizing effect due to the direct cholesterol-peptide interaction with the involvement of CRAC1 (L14-K23) and/or CCM (K40-W50) in amyloidogenic peptide 1-93 is suggested to underlie the known effect of cholesterol on the induction of helix formation in this peptide. Cholesterol may be speculated to act in helix formation as a chemical chaperone thus triggering disorder-to-order transition within MoRF; without cholesterol, this MoRF may be involved into protein-protein interaction. Indeed, the association of W50R substitution with the accelerated mutant aggregation rate and the involvement of L44-S55 segment into cross-beta-sheet formation were demonstrated by others. Also, D102H substitution in CRAC2 (V97-K106) may be harmful in RCT. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in CRAC2 motif of apoA-I in nascent HDL. Besides, the resistance of two-domain apoA-I structure to denaturation by GuHCl is sensitive to cholesterol level in discoidal HDL. Overall, different CRACs may contribute into amyloidosis-connected apoA-I misfolding and cholesterol efflux for lipid-free apoA-I. Cholesterol binding to lipid-bound apolipoprotein may change the structural plasticity of two domains and/or their relative dimensions.

Lipid-bound apolipoproteins in tyrosyl radical-oxidized HDL stabilize ABCA1 like lipid-free apolipoprotein A-I

BackgroundATP-binding cassette transporter A1 (ABCA1) mediates the lipidation of exchangeable apolipoproteins, the rate-limiting step in the formation of high density lipoproteins (HDL). We previously demonstrated that HDL oxidized ex vivo by peroxidase-generated tyrosyl radical (tyrosylated HDL, tyrHDL) increases the availability of cellular cholesterol for efflux and reduces the development of atherosclerosis when administered to apolipoprotein E-deficient mice as compared to treatment with control HDL.ResultsIn the current study we determined that tyrHDL requires functional ABCA1 for this enhanced activity. Like lipid-free apolipoprotein A-I (apoA-I), tyrHDL increases total and cell surface ABCA1, inhibits calpain-dependent and -independent proteolysis of ABCA1, and can be bound by cell surface ABCA1 in human skin fibroblasts. Additionally, tyrHDL apoproteins are susceptible to digestion by enteropeptidase like lipid-free apoA-I, but unlike lipid-bound apoA-I on HDL, which is resistant to proteolysis.ConclusionsThese results provide the first evidence that lipid-bound apolipoproteins on the surface of spherical HDL particles can behave like lipid-free apoA-I to increase ABCA1 protein levels and activity.

Analysis of apolipoprotein A-I as a substrate for matrix metalloproteinase-14

Substrates for matrix metalloproteinase (MMP)-14 were previously identified in human plasma using proteomic techniques. One putative MMP-14 substrate was apolipoprotein A-I (apoA-I), a major component of high-density lipoprotein (HDL). In vitro cleavage assays showed that lipid-free apoA-I is a more accessible substrate for MMP-14 compared to lipid-bound apoA-I, and that MMP-14 is more prone to digest apoA-I than MMP-3. The 28-kDa apoA-I was cleaved into smaller fragments of 27, 26, 25, 22, and 14-kDa by MMP-14. ApoA-I sites cleaved by MMP-14 were determined by isotope labeling of C-termini derived from the cleavage and analysis of the labeled peptides by mass spectrometry, along with N-terminal sequencing of the fragments. Cleavage of apoA-I by MMP-14 resulted in a loss of ability to form HDL. Our results suggest that cleavage of lipid-free apoA-I by MMP-14 may contribute to reduced HDL formation, and this may be occurring during the development of various vascular diseases as lipid metabolism is disrupted.

Structure of Apolipoprotein A-I N Terminus on Nascent High Density Lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.

Anti-inflammatory action of lipid-bound apoA-I cysteine mutants

To determine the anti-inflammatory functions of different cysteine mutants of apolipoprotein A-I recombinant HDLs. The recombinant HDLs (named rHDL52, rHDL107, rHDL173, rHDLwt) were reconstituted by mixing wild types or their mutants with dipalmitoyl phosphatidylcholine. And the in vivo effects on lipopolysaccharide (LPS)-induced endotoxemia were examined in mice. The plasma levels of plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in by ELISA were tested. And we also set up two control groups: LPS and saline. The rHDL52 mice had a significant decrease of plasma TNF-alpha and IL-1beta and the protection of lung against acute injury. 24 h post-injection as compared with rHDLwt group [TNF-alpha: (135.28 +/- 12.84) pg/ml, IL-1beta: (82.00 +/- 8.19) pg/ml], the rHDL52 mice exhibited a higher capability of lowering the plasma levels of TNF-alpha and IL-1beta [(39.66 +/- 2.44) pg/ml, (66.83 +/- 6.24) pg/ml, both P < 0.05]. And, as indicated by histological sections of lung tissue, the rHDL52 mice could also lower the infiltration of inflammatory cells in lung. rHDL52 has a higher anti-inflammation capability than wild type rHDLwt.

Effect of lipid-bound apoA-I cysteine mutants upon lipopolysaccharide-induced endotoxemia in mice

To determine the anti-inflammatory functions of different cysteine mutants of apolipoprotein A-I recombinant HDLs. The authors reconstituted recombinant HDLs (namely rHDL74, rHDL129, rHDL195 and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects upon LPS-induced endotoxemia in mice. At 24 h post-injection, mice receiving rHDL74 [TNF-alpha: (24 +/- 3) pg/ml; IL-1beta: (45 +/- 5) pg/ml] had a significant decrease of plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) as compared with control mice receiving either saline or rHDLwt [TNF-alpha: (135 +/- 12) pg/ml; IL-1beta: (82 +/- 8) pg/ml, P < 0.05]. Administration of rHDL74 to mice injected with LPS also led to a protection of lung against acute injury and attenuation of endotoxin-induced clinical symptoms in mice as compared with controls injected with LPS only. Compared with rHDLwt, rHDL74 exhibits higher anti-inflammation capabilities. And it may be a potential clinical candidate for therapy for endotoxin-induced septic shock.

Apolipophorin III from Hyphantria cunea shows different anti-oxidant ability against LDL oxidation in the lipid-free and lipid-bound state

Insect apolipophorin III (apoLp-III) and human apolipoprotein A-I (apoA-I) are major protein constituents of the lipoprotein system that share various properties. In order to compare the anti-oxidant ability of apoLp-III and apoA-I in the lipid-free and lipid-bound state, both proteins were purified and synthesized individually as a palmitoyloleoyl phosphatidylcholine (POPC)-reconstituted high-density lipoprotein (rHDL) using the same molar ratio. In the lipid-bound state, apoLp-III and apoA-I showed good anti-oxidant activities against copper-mediated LDL oxidation. Furthermore, apoLp-III and apoA-I, in the lipid-bound state, exhibited potent activities in ferric ion-reducing ability (FRA). However, lipid-free apoLp-III lost the anti-oxidant activity and FRA ability in contrast to lipid-free apoA-I. Lipid-free apoA-I treatment prevented the cellular uptake of oxLDL in macrophages, as visualized by oil-red O staining and detection assays for malondialdehyde (MDA) and lipid hydroperoxide (LPO) in the culture media. However, lipid-free apoLp-III did not prevent the uptake of oxLDL. These results indicate that the putative conformational change of apoLp-III during lipid association is critical for the maintenance of anti-oxidant activity and that the physiologic role of apoLp-III may differ when it is in the lipid-free state and the lipid-bound state.

Effect of lipid-bound apoA-I cysteine mutants on lipopolysaccharide-induced endotoxemia in mice

HDL has been shown to be able to neutralize the toxicity of lipopolysaccharide (LPS). Our previous study (J. Lipid Res. 2005. 46: 1303-1311) characterized the properties of secondary structure and in vitro functions of different cysteine mutants of apolipoprotein A-I. Here, we reconstituted recombinant HDLs (named rHDLwt, rHDL52, rHDL74, rHDL107, rHDL129, rHDL173, rHDL195, and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects on LPS-induced endotoxemia in mice. Our results showed that 24 h after injection, mice receiving rHDL74 or rHDL52 had a significant decrease of plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), compared with control mice receiving either saline or rHDLwt (P < 0.05). Administration of rHDL74 to mice injected with LPS also led to a decrease of plasma IL-6, protection of lung against acute injury, and attenuation of endotoxin-induced clinical symptoms in mice, compared with controls injected with LPS only. However, injection of rHDL228 significantly increased plasma concentration of TNF-alpha and exacerbated LPS-induced lung injury. In summary, compared with rHDLwt, rHDL74 and rHDL52 exhibit higher anti-inflammation capabilities, whereas rHDL228 shows hyper-proinflammation by exacerbating LPS-induced endotoxemia in mice.

Conformational adaptation of apolipoprotein A-I to discretely sized phospholipid complexes.

The conformational constraints for apoA-I bound to recombinant phospholipid complexes (rHDL) were attained from a combination of chemical cross-linking and mass spectrometry. Molecular distances were then used to refine models of lipid-bound apoA-I on both 80 and 96 A diameter rHDL particles. To obtain molecular constraints on the protein bound to phospholipid complexes, three different lysine-selective homo-bifunctional cross-linkers with increasing spacer arm lengths (i.e., 7.7, 12.0, and 16.1 A) were reacted with purified, homogeneous recombinant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) apoA-I rHDL complexes of each diameter. Cross-linked dimeric apoA-I products were separated from monomeric apoprotein using 12% SDS-PAGE, then subjected to in-gel trypsin digest, and identified by MS/MS sequencing. These studies aid in the refinement of our previously published molecular model of two apoA-I molecules bound to approximately 150 molecules of POPC and suggest that the protein hydrophobic interactions at the N- and C-terminal domains decrease as the number of phospholipid molecules or "lipidation state" of apoA-I increases. Thus, it appears that these incremental changes in the interaction between the N- and C-terminal ends of apoA-I stabilize its tertiary conformation in the lipid-free state as well as allowing it to unfold and sequester discrete amounts of phospholipid molecules.

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Role of apoA-I, ABCA1, LCAT, and SR-BI in the biogenesis of HDL

The concentration, composition, shape, and size of plasma high-density lipoprotein (HDL) are determined by numerous proteins that influence its biogenesis, remodeling, and catabolism. The discoveries of the HDL receptor (scavenger receptor class B type I, SR-BI) and the ABCA1 (ATP-binding cassette transporter A1) lipid transporter provided two missing links that were necessary to understand the biogenesis and some of the functions of HDL. Existing data indicate that functional interactions between apoA-I and ABCA1 are necessary for the initial lipidation of apoA-I. Through a series of intermediate steps, lipidated apoA-I proceeds to form discoidal HDL particles that can be converted to spherical particles by the action of lecithin:cholesterol acyltransferase (LCAT). Discoidal and spherical HDL can interact functionally with SR-BI and these interactions lead to selective lipid uptake and net efflux of cholesterol and thus remodel HDL. Defective apoA-I/ABCA1 interactions prevent lipidation of apoA-I that is necessary for the formation of HDL particles. In the same way, specific mutations in apoA-I or LCAT prevent the conversion of discoidal to spherical HDL particles. The interactions of lipid-bound apoA-I with SR-BI are affected in vitro by specific mutations in apoA-I or SR-BI. Furthermore, deficiency of SR-BI affects the lipid and apolipoprotein composition of HDL and is associated with increased susceptibility to atherosclerosis. Here we review the current status of the pathway of HDL biogenesis and mutations in apoA-I, ABCA1, and SR-BI that disrupt different steps of the pathway and may lead to dyslipidemia and atherosclerosis in mouse models. The phenotypes generated in experimental mouse models for apoA-I, ABCA1, LCAT, SR-BI, and other proteins of the HDL pathway may facilitate early diagnosis of similar phenotypes in the human population and provide guidance for proper treatment.

Intermolecular Contact between Globular N-terminal Fold and C-terminal Domain of ApoA-I Stabilizes Its Lipid-bound Conformation: STUDIES EMPLOYING CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY

The structure of apoA-I on discoidal high density lipoprotein (HDL) was studied using a combination of chemical cross-linking and mass spectrometry. Recombinant HDL particles containing 145 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and two molecules of apoA-I with a 96-A diameter were treated with the lysine-specific cross-linker, dithiobis(succinimidylpropionate) at varying molar ratios from 2:1 to 200:1. At low molar ratios of dithiobis(succinimidylpropionate) to apoA-I, two products were obtained corresponding to approximately 53 and approximately 80 kDa. At high molar ratios, these two products merged, yielding a product of approximately 59 kDa, close to the theoretical molecular mass of dimeric apoA-I. To identify the intermolecular cross-links giving rise to the two different sized products, bands were excised from the gel, digested with trypsin, and then analyzed by liquid chromatography-electrospray-tandem mass spectrometry. In addition, tandem mass spectrometry of unique cross-links found in the 53- and 80-kDa products suggested that a distinct conformation exists for lipid-bound apoA-I on 96-A recombinant HDL, emphasizing the inherent flexibility and malleability of the N termini and its interaction with its C-terminal domain.

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.

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.

Cholesterol mobilization by free and lipid-bound apoAI(Milano) and apoAI(Milano)-apoAII heterodimers.

Despite very low plasma levels of HDL, carriers of the apolipoprotein AI Arg173 --> Cys mutation apoAI(Milano) (AIM) have no apparent increase in risk for atherosclerotic vascular disease. HDL apolipoprotein species in AIM carriers include apoAI-AII heterodimers, previously found to confer the enhanced ability of tyrosyl radical-oxidized HDL to mobilize cholesterol for removal from cultured cells. To determine whether enhanced mobilization of cholesterol by apoprotein species in AIM explains a cardioprotective action of this mutation, we examined the ability of lipid-free and lipid-bound AIM and AIM-AII heterodimers to deplete cholesterol from cultured cells. Free AIM and AIM-AII heterodimers showed a decreased capacity to act as acceptors of cholesterol from cholesterol-loaded human fibroblasts compared with native apoAI but similar capacities to deplete fibroblasts of the pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Discoidal reconstituted HDL (rHDL) containing apoAI depleted both of these cholesterol pools more readily than AIM-containing rHDL when compared at equivalent rHDL protein levels, but similar abilities of these rHDL to deplete cell cholesterol were seen when compared at equivalent phospholipid levels. Spherical rHDL generated using the whole lipid fraction of HDL and apoAI or AIM showed similar capacities to deplete total and ACAT-accessible cell cholesterol when compared at similar protein levels, but an increased capacity of AIM-containing particles was seen when compared at equivalent phospholipid levels. Unlike the apoAI-AII heterodimer in tyrosylated HDL, AIM-AII heterodimer-containing spherical rHDL showed no increased capacity to deplete either of these pools of cholesterol. These results suggest a similar or better capacity of native apoAI in lipid-free or lipid-bound form in discoidal rHDL to enhance the mobilization of cellular cholesterol when compared to AIM in its free or lipid-bound forms. Any increase in depletion of cellular cholesterol by lipid-bound AIM in spherical rHDL appears related to altered phospholipid-binding rather than intrinsic cholesterol-mobilizing characteristics of this protein compared to native apoAI. The lack of major differences in these studies in cholesterol mobilization by native apoAI and AIM, or by apoAIM-AII heterodimers, suggests that any protection against atherosclerosis conferred by this mutation is likely related to other beneficial vascular effects of AIM.

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.