Identification of a sequence of apolipoprotein A-I associated with the activation of Lecithin:Cholesterol acyltransferase

We aimed to distinguish between the effects of mutations in apoA-I on the requirements for the secondary structure and a specific amino acid sequence for lecithin:cholesterol acyltransferase (LCAT) activation. Several mutants were constructed targeting region 140-150: (i) two mutations affecting alpha-helical structure, deletion of amino acids 140-150 and substitution of Ala(143) for proline; (ii) two mutations not affecting alpha-helical structure, substitution of Val(149) for arginine and substitution of amino acids 63-73 for sequence 140-150; and (iii) a mutation in a similar region away from the target area, deletion of amino acids 63-73. All mutations affecting region 140-150 resulted in a 4-42-fold reduction in LCAT activation. Three mutations, apoA-I(Delta140-150), apoA-I(P143A), and apoA-I(140-150 --> 63-73), affected both the apparent V(max) and K(m), whereas the mutation apoA-I(R149V) affected only the V(max). The mutation apoA-I(Delta63-73) caused only a 5-fold increase in the K(m). All mutants, except apoA-I(P143A) and apoA-I(Delta63-73), were active in phospholipid binding assay. All mutants, except apoA-I(P143A), formed normal discoidal complexes with phospholipid. The mutation apoA-I(Delta63-73) caused a significant reduction in the stability of apoA-I.phospholipid complexes in denaturation experiments. Combined, our results strongly suggest that although the correct conformation and orientation of apoA-I in the complex with lipids are crucial for activation of LCAT, when these conditions are fulfilled, activation also strongly depends on the sequence that includes amino acids 140-150.     

Effectivity of expression of mature forms of mutant human apolipoprotein A-I.

In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.     

Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage.

The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.     

The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.     

Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1

To explore the functional interactions between apoA-I and ABCA1, we correlated the cross-linking properties of several apoA-I mutants with their ability to promote cholesterol efflux. In a competitive cross-linking assay, amino-terminal deletion and double amino- and carboxy-terminal deletion mutants of apoA-I competed effectively the cross-linking of WT (125)I-apoA-I to ABCA1, while the carboxy-terminal deletion mutant apoA-I[Delta(220-243)] competed poorly. Direct cross-linking of WT apoA-I, amino-terminal, and double deletion mutants of apoA-I to ABCA1 showed similar apparent K(d) values (49-74 nM), whereas the apparent K(d) values of the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] were increased 3-fold. Analysis of several internal deletions and point mutants of apoA-I showed that apoA-I[Delta(61-78)], apoA-I[Delta(89-99)], apoA-I[Delta(136-143)], apoA-I[Delta(144-165)], apoA-I[D102A/D103A], apoA-I[E125K/E128K/K133E/E139K], apoA-I[L141R], apoA-I[R160V/H162A], and WT apoA-I had similar ABCA1-mediated lipid efflux, and all competed efficiently the cross-linking of WT (125)I-apoA-I to ABCA1. WT apoA-I and ABCA1 could be cross-linked with a 3 A cross-linker. The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. The findings are consistent with a direct association of different combinations of apoA-I helices with a complementary ABCA1 domain. Mutations that alter ABCA1/apoA-I association affect cholesterol efflux and inhibit biogenesis of HDL.     

Structure and stability of apolipoprotein a-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Delta(61-78) and Delta(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid-cholesterol-apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4 and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation, suggesting involvement of R160/H162 in important interhelical interactions. The results on the Delta(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by specific electrostatic interhelical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.     

Distinct central amphipathic alpha-helices in apolipoprotein A-I contribute to the in vivo maturation of high density lipoprotein by either activating lecithin-cholesterol acyltransferase or binding lipids

Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Delta4-5A-I, Delta5-6A-I, and Delta6-7A-I, that have deletions removing regions coding for amino acids 100-143, 122-165, and 144-186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Delta5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Delta4-5A-I and Delta6-7A-I that formed more buoyant HDL(2/3) particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Delta5-6A-I and Delta6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Delta4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Delta6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Delta5-6A-I and greatly for Delta4-5A-I. We conclude from these findings that amino acids (aa) 100-143, specifically helix 4 (aa 100-121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144-186) centered around helix 6 (aa 144-165) is responsible for the activation of lecithin-cholesterol acyltransferase.     

Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements

The central region of apolipoprotein A-I (apoA-I), spanning residues 143--165, has been implicated in lecithin:cholesterol acyltransferase (LCAT) activation and also in high density lipoprotein (HDL) structural rearrangements. To examine the role of individual amino acids in these functions, we constructed, overexpressed, and purified two additional point mutants of apoA-I (P143R and R160L) and compared them with the previously studied V156E mutant. These mutants have been reported to occur naturally and to affect HDL cholesterol levels and cholesterol esterification in plasma. The P143R and R160L mutants were effectively expressed in Escherichia coli as fusion proteins and were isolated in at least 95% purity. In the lipid-free state, the mutants self-associated similarly to wild-type protein. All the mutants, including V156E, were able to lyse dimyristoylphosphatidylcholine liposomes. In the lipid-bound state, the major reconstituted HDL (rHDL) of the mutants had diameters similar to wild type (96--98 A). Circular dichroism and fluorescence methods revealed no major differences among the structures of the lipid-free or lipid-bound mutants and wild type. In contrast, the V156E mutant had exhibited significant structural, stability, and self-association differences compared with wild-type apoA-I in the lipid-free state, and formed rHDL particles with larger diameters. In this study, limited proteolytic digestion with chymotrypsin showed that the V156E mutant, in lipid-free form, has a distinct digestion pattern and surface exposure of the central region, compared with wild type and the other mutants. Reactivity of rHDL with LCAT was highest for wild type (100%), followed by P143R (39%) and R160L (0.6%). Tested for their ability to rearrange into 78-A particles, the rHDL of the two mutants (P143R and R160L) behaved normally, compared with the rHDL of V156E, which showed no rearrangement after the 24-h incubation with low density lipoprotein (LDL). Similarly, the rHDL of V156E was resistant to rearrangement in the presence of apoA-I or apoA-II. These results indicate that structural changes are absent or modest for the P143R and R160L mutants, especially in rHDL form; that these mutants have normal conformational adaptability; and that LCAT activation is obliterated for R160L.Thus, individual amino acid changes may have markedly different structural and functional consequences in the 143--165 region of apoA-I. The R160L mutation appears to have a direct effect in LCAT activation, while the P143R mutation results in only minor structural and functional effects. Also, the processes for LCAT activation and hinge mobility appear to be distinct even if the same region of apoA-I is involved. -- Cho, K-H., D. M. Durbin, and A. Jonas. Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements. J. Lipid Res. 2001. 42: 379--389.     

Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69–99% of control by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.     

Lipid-binding studies of human apolipoprotein A-I and its terminally truncated mutants

Apolipoprotein A-I (apoA-I, 243 amino acids) is the major protein of high-density lipoproteins (HDL) that plays an important structural and functional role in lipid transport and metabolism. The central region of apoA-I (residues 60-183) is predicted to contain exclusively amphipathic alpha-helices formed from tandem 22-mer sequence repeats. To analyze the lipid-binding properties of this core domain, four terminally truncated mutants of apoA-I, Delta(1-41), Delta(1-59), Delta(1-41,185-243), and Delta(1-59,185-243), were expressed in baculovirus infected Sf-9 cells. The effects of mutations on the ability of apoA-I to form bilayer disk complexes with dimyristoyl phosphatidylcholine (DMPC) that resemble nascent HDL were analyzed by density gradient ultracentrifugation and electron microscopy (EM). The N-terminal deletion mutants, Delta(1-41) and Delta(1-59), showed altered lipid-binding ability as compared to plasma and wild-type apoA-I, and in the double deletion mutants, Delta(1-41, 185-243) and Delta(1-59, 185-243), the lipid binding was abolished. Thermal unfolding of variant apoA-I/DMPC complexes monitored by circular dichroism (CD) showed hysteresis and a shift in the melting curves by about -12 degrees C upon reduction in the heating rate from 1.0 to 0.067 K/min. This indicates an irreversible kinetically controlled transition with a high activation energy E(a) = 60 +/- 5 kcal/mol. CD and EM studies of the apoA-I/DMPC complexes at different pH demonstrated that changes in the net charge or in the charge distribution on the apoA-I molecule have critical effects on the conformation and lipid-binding ability of the protein.     

Characterization and properties of pre beta-HDL particles formed by ABCA1-mediated cellular lipid efflux to apoA-I

The contribution of ABCA1-mediated efflux of cellular phospholipid (PL) and cholesterol to human apolipoprotein A-I (apoA-I) to the formation of pre beta 1-HDL (or lipid-poor apoA-I) is not well defined. To explore this issue, we characterized the nascent HDL particles formed when lipid-free apoA-I was incubated with fibroblasts in which expression of the ABCA1 was upregulated. After a 2 h incubation, the extracellular medium contained small apoA-I/PL particles (pre beta 1-HDL; diameter = 7.5 +/- 0.4 nm). The pre beta 1-HDL (or lipid-poor apoA-I) particles contained a single apoA-I molecule and three to four PL molecules and one to two cholesterol molecules. An apoA-I variant lacking the C-terminal alpha-helix did not form such particles when incubated with the cell, indicating that this helix is critical for the formation of lipid-poor apoA-I particles. These pre beta 1-HDL particles were as effective as lipid-free apoA-I molecules in mediating both the efflux of cellular lipids via ABCA1 and the formation of larger, discoidal HDL particles. In conclusion, pre beta 1-HDL is both a product and a substrate in the ABCA1-mediated reaction to efflux cellular PL and cholesterol to apoA-I. A monomeric apoA-I molecule associated with three to four PL molecules (i.e., lipid-poor apoA-I) has similar properties to the lipid-free apoA-I molecule.     

Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids

The influence of apolipoprotein (apo) A-I structure on ABCA1-mediated efflux of cellular unesterified (free) cholesterol (FC) and phospholipid (PL) is not well understood. To address this issue, we used a series of apoA-I mutants to examine the contributions of various domains in the molecule to ABCA1-mediated FC and PL efflux from mouse J774 macrophages and human skin fibroblasts. Irrespective of the cell type, deletion or disruption of the C-terminal lipid-binding domain of apoA-I drastically reduced the FC and PL efflux ( approximately 90%), indicating that the C-terminal amphipathic alpha-helix is required for high affinity microsolubilization of FC and PL. Deletion in the N-terminal region of apoA-I also reduced the lipid efflux ( approximately 30%) and increased the K(m) about 2-fold compared with wild type apoA-I, whereas deletion of the central domain (Delta123-166) had no effect on either K(m) or V(max). These results indicate that ABCA1-mediated lipid efflux is relatively insensitive to the organization of the apoA-I N-terminal helix-bundle domain. Alterations in apoA-I structure caused parallel changes in its ability to bind to a PL bilayer and to induce efflux of FC and PL. Overall, these results are consistent with a two-step model for ABCA1-mediated lipid efflux. In the first step, apoA-I binds to ABCA1 and hydrophobic alpha-helices in the C-terminal domain of apoA-I insert into the region of the perturbed PL bilayer created by the PL transport activity of ABCA1, thereby allowing the second step of lipidation of apoA-I and formation of nascent high density lipoprotein particles to occur.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

Tyrosine 192 in apolipoprotein A-I is the major site of nitration and chlorination by myeloperoxidase, but only chlorination markedly impairs ABCA1-dependent cholesterol transport

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO(-)). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO(-). Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr(192) of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO(-), suggesting that solvent accessibility accounted in part for the reactivity of Tyr(192). The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) is the predominant site of nitration and chlorination when MPO or ONOO(-) oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

Structural and functional basis for increased HDL-cholesterol levels due to the naturally occurring V19L mutation in human apolipoprotein A-I

Several hereditary point mutations in human apolipoprotein A-I (apoA-I) have been associated with low HDL-cholesterol levels and/or increased coronary artery disease (CAD) risk. However, one apoA-I mutation, the V19L, recently identified in Icelanders, has been associated with increased HDL-cholesterol levels and decreased CAD risk. In an effort to gain mechanistic insight linking the presence of this mutation in apoA-I with the increase of HDL-cholesterol levels we evaluated the effect of V19L mutation on the conformational integrity and functional properties of apoA-I in lipid-free and lipidated form. ApoA-I[V19L] was found to be thermodynamically destabilized in lipid-free form and displays an increased capacity to associate with phospholipids compared to WT apoA-I. When associated to reconstituted HDL (rHDL), apoA-I[V19L] was more thermodynamically stabilized than WT apoA-I. ApoA-I[V19L] displayed normal capacity to promote ABCA1-mediated cholesterol efflux and to activate the enzyme LCAT, in lipid-free and rHDL-associated forms, respectively. Additionally, rHDL-associated apoA-I[V19L] showed normal capacity to promote ABCG1-mediated cholesterol efflux, but 45% increased capacity to promote SR-BI-mediated cholesterol efflux, while the SR-BI-mediated HDL-lipid uptake was normal. Overall, our findings show that the apoA-I V19L mutation does not affect the first steps of HDL biogenesis pathway. However, the increased capacity of apoA-I[V19L] to associate with phospholipids, in combination with the enhanced thermodynamic stability of lipoprotein-associated apoA-I[V19L] and increased capacity of apoA-I[V19L]-containing lipoprotein particles to accept additional cholesterol by SR-BI could account for the increased HDL-cholesterol levels observed in human carriers of the mutation.     

Substitutions of glutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanine affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia in apoA-I-deficient mice

Hypertriglyceridemia is a common pathological condition in humans of mostly unknown etiology. Here we report induction of dyslipidemia characterized by severe hypertriglyceridemia as a result of point mutations in human apolipoprotein A-I (apoA-I). Adenovirus-mediated gene transfer in apoA-I-deficient (apoA-I(-)(/)(-)) mice showed that mice expressing an apoA-I[E110A/E111A] mutant had comparable hepatic mRNA levels with WT controls but greatly increased plasma triglyceride and elevated plasma cholesterol levels. In addition, they had decreased apoE and apoCII levels and increased apoB48 levels in very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL). Fast protein liquid chromatography (FPLC) analysis of plasma showed that most of cholesterol and approximately 15% of the mutant apoA-I were distributed in the VLDL and IDL regions and all the triglycerides in the VLDL region. Hypertriglyceridemia was corrected by coinfection of mice with recombinant adenoviruses expressing the mutant apoA-I and human lipoprotein lipase. Physicochemical studies indicated that the apoA-I mutation decreased the alpha-helical content, the stability, and the unfolding cooperativity of both lipid-free and lipid-bound apoA-I. In vitro functional analyses showed that reconstituted HDL (rHDL) particles containing the mutant apoA-I had 53% of scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacity and 37% capacity to activate lecithin:cholesterol acyltransferase (LCAT) as compared to the WT control. The mutant lipid-free apoA-I had normal capacity to promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. The findings indicate that subtle structural alterations in apoA-I may alter the stability and functions of apoA-I and high-density lipoprotein (HDL) and may cause hypertriglyceridemia.     

Oxysterol efflux from macrophage foam cells: the essential role of acceptor phospholipid.

Oxidized forms of cholesterol (oxysterols) are present in atherosclerotic lesions and may play an active role in lesion development. For example, 7-ketocholesterol (7KC) inhibits cholesterol efflux from macrophage foam cells induced by apolipoprotein A-I (apoA-I). Such oxysterols may promote foam cell formation in atherosclerotic lesions by preventing effective clearance of excess cholesterol. ApoA-I also induces phospholipid (PL) export from foam cells and it has been suggested that cholesterol efflux is dependent upon PL association with the apolipoprotein. In the current study, the effect of oxysterol enrichment of foam cells on phospholipid efflux was measured. Export of cellular PL to apoA-I from 7KC-enriched foam cells was inhibited to the same extent as cholesterol, indicating that the reduced cholesterol export may be a consequence of a decline in the capacity of the foam cells to generate PL/apoA-I particles capable of accepting cellular cholesterol. Incubation of foam cells with pre-formed PL/apoA-I discs increased cholesterol export from 7KC-enriched cells to levels seen in 7KC-free cells. Foam cells produced by uptake of oxidized LDL, which contain similar amounts of 7KC plus other oxidation products, expressed a more profound inhibition of PL export to apoA-I. Cholesterol efflux from these cells improved only partially by provision of PL-containing acceptors. Efflux of 7KC from both foam cell types occurred to PL/apoA-I discs but was only minimal to lipid-free apoA-I, indicating that export of this oxysterol is more dependent than cholesterol upon the presence of extracellular phospholipid.