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J. Biol. Chem., Vol. 279, Issue 23, 24044-24052, June 4, 2004
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From the
Lawrence Berkeley National Laboratory, Donner Laboratory MS1224, University of California at Berkeley, California 94720,
Lipid Research Group, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, and ¶Department of Medicine, Endocrinology, and Nutrition, University of Washington School of Medicine, Seattle, Washington 98195-6426
Received for publication, January 20, 2004 , and in revised form, March 11, 2004.
| ABSTRACT |
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-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic
-helices is required to mediate cholesterol efflux and stabilize ABCA1. | INTRODUCTION |
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The ATP binding cassette transporter A1 (ABCA1) is defective in Tangier disease (8, 9). Mutations in ABCA1 abolish the ability of apoA-I to mediate phospholipid and cholesterol efflux from cells producing HDL deficiency and premature atherosclerosis (813). Targeted disruption of ABCA1 in mice produces a phenotype similar to human Tangier disease (14, 15), whereas overexpression of ABCA1 protects against atherosclerosis in mice (16, 17). These findings underscore the importance of apoA-I/ABCA1 interactions in heart disease protection. ApoA-I has been shown to stabilize ABCA1 in the plasma membrane, preventing its rapid degradation (1820). Stabilizing ABCA1 represents a potential target of therapeutic interventions for up-regulating cellular ABCA1 protein and optimizing cholesterol efflux.
Despite the great importance of ABCA1 in HDL biogenesis and atherosclerosis protection, very little is known about the structural determinants of apoA-I that endow cholesterol efflux and ABCA1 stabilization activities. Apolipoprotein A-I is a 243-amino acid protein possessing distinct structural domains (21, 22). The C-terminal domain (aa 44243) of apoA-I is able to mediate cholesterol efflux, indicating that it contains the helical segments responsible for interacting with the ABCA1 transporter (23, 24). The amphipathic
-helices that define the architecture of the C-terminal domain of apoA-I consist of 11 and 22 amino acids arranged in series and separated by proline residues (21, 25). The 11-mer helical segment represents the smallest unit of
-helix, forming three complete turns of secondary structure, whereas the 22-mer helix probably emerged via duplication events within the apoA-I gene (21). The amphipathic
-helical segments of apoA-I are classified as either class A or Y based on the distribution of positively and negatively charged amino acids on the polar surface (21).
Helices 1 (aa 4465) and 10 (aa 220241) of apoA-I have been implicated as mediators of cellular lipid efflux because these segments possess the highest lipid binding affinity as synthetic 22-mer peptides compared with the other amphipathic
-helices of the C-terminal domain (26, 27). Despite the fact that helices 1 and 10 both possess relatively high lipid binding affinity, only helix 1 is able to stimulate cholesterol efflux, whereas helix 10 cannot unless joined to helix 9, as judged from efflux studies utilizing cholesterol-loaded fibroblasts (27). Gillotte et al. (27) demonstrate that helix 1 (22-mer peptide) and the 9/10 combination (33-mer) were
50 and 68% as effective as full-length apoA-I in mediating cellular cholesterol efflux, respectively.
It is currently not known whether individual amphipathic
-helices derived from apoA-I mediate cholesterol efflux via ABCA1 and/or if multiple
-helices in tandem are required. The helical segments of apoA-I that stabilize ABCA1 protein are also not known. Thus, we sought to identify the minimum helical structure of apoA-I that was sufficient to mediate cellular cholesterol efflux and stabilize ABCA1. We found that the relatively high lipid binding affinity of helices 1 and 10 alone was not sufficient to mediate cholesterol efflux via ABCA1. The joining of two complementary amphipathic
-helices is required to create a specific structural element with both cholesterol efflux and ABCA1 stabilization properties.
| EXPERIMENTAL PROCEDURES |
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-helical segments. The following list defines the amino acid segments used to create synthetic peptides including individual 11- and 22-mer helices, unique chimeras, native helical combinations, and transposition peptides: helix 1 peptide, aa 4465 (22-mer); helix 9, aa 209219 (11-mer); helix 10, aa 220241 (22-mer); 1/9 chimera, aa 4465/209219 (33-mer); 1/3 chimera, aa 4465/8898 (33-mer); 2/9 chimera, aa 6687/209219 (33-mer); 4/9 chimera, aa 99120/209219 (33-mer); the 9/10 peptide, aa 209241 (33-mer); 10/9 transposition peptide, aa 220241/209219 (33-mer), and 9/1, aa 209219/4465 (33-mer). Biosynthesis Inc (Lewisville, TX) synthesized the peptides. All peptides were isolated by high performance liquid chromatography and used at a purity of
95%. The peptides were synthesized with an N-terminal acetyl group and a C-terminal amide to stabilize the amphipathic
-helices (28). Stock solutions (0.51 mg/ml) were prepared by dissolving the lyophilized peptides in sterile Tris-HCl (10 mM) buffered (pH 7.4) saline and stored at 4 °C. Protein concentrations were set by the mass data provided by the manufacturer and were verified using a BCA reagent kit (Pierce).
Apolipoprotein A-IA bacterial expression system was used to generate full-length apoA-I as previously described (29, 30) using a histidine (His) tag to facilitate protein purification. The purified recombinant protein was
98% pure and exhibited a molecular mass of 28 kDa, similar to native apoA-I purified from human plasma (29, 30). Control experiments verified that the recombinant apoA-I behaved exactly the same as native apoA-I with regard to mediating cholesterol efflux in an ABCA1-dependent manner (data not shown). The present study was conducted using His-tagged apoA-I, which exhibits normal cholesterol efflux capability similar to apoA-I without a His tag (data not shown).
Cellular Cholesterol Efflux ProtocolJ774 macrophages were used to assess the cholesterol efflux properties of synthetic amphipathic
-helical peptides (30, 31). This cell line was chosen because cholesterol efflux can be enhanced using a cAMP analog that up-regulates ABCA1 protein expression. The cells were seeded onto 24-well culture plates and labeled for 48 h with [3H]cholesterol in RPMI 1640 supplemented with 1% fetal bovine serum. The cAMP analog CPT-cAMP was added (0.3 mM, final concentration) to the cells at least 12 h before the initiation of cellular cholesterol efflux. Synthetic peptides in lipid-free form were added to cells in serum-free RPMI. The lipid-free form of full-length recombinant apoA-I was used as a positive control to define apparent ABCA1-dependent cholesterol efflux in the presence and absence of cAMP stimulation. Efflux results were expressed as a percentage of the initial cellular [3H] appearing in the medium as a function of time subtracting the background efflux obtained using serum-free medium alone.
Relative Lipid Binding Affinity, Hydrophobicity, and Amphiphilicity of Synthetic PeptidesIn some experiments, the relative lipid binding affinity of unique peptides was quantified using a surface balance technique (27). For routine analyses, a turbid solution of dimyristoylphosphatidylcholine (DMPC) was used to assess the relative capacity of synthetic peptides to solubilize phospholipid as described (26, 32). The DMPC was used at a final concentration of 0.08 mg/ml in 10 mM Tris-saline (pH 7.4). The final weight ratio of peptides relative to DMPC was 1:1. The absorbance (400 nm) of samples was monitored continuously over a period of 30 min at 25 °C. Hydrophobicity of helical peptides was calculated using the consensus scale (33). The hydrophobic moment (kcal/mol) of synthetic peptides, which is a measure of helix amphiphilicity, was calculated as described by Eisenberg et al. (34).
ABCA1 StabilizationJ774 macrophages were used to assess relative ABCA1 protein levels in the presence and absence of synthetic peptides. Cells were grown in 10% fetal bovine serum, extensively rinsed, and incubated (18 h) with Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin plus the cAMP analog 8-bromo-cAMP. Cells were next exposed to synthetic peptides or serum-free medium in the absence of the cAMP stimulus to evaluate whether ABCA1 protein was stabilized or degraded. Relative levels of ABCA1 protein were assessed by immunoblot analysis of cell membranes (35). ABCA1 was visualized using an enhanced chemiluminescence detection assay.
| RESULTS |
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-helices in tandem may be required to create a structural element that stimulates cellular cholesterol efflux via the ABCA1 transporter. Thus, we sought to identify the minimum sequence requirements that endowed cholesterol efflux capability. To this end, we asked whether the addition of the 11-mer helix 9 (aa 209219) to helix 1 produced an increase in cholesterol efflux. A 33-mer peptide composed of helices 1 plus 9 (1/9 chimera) stimulated cholesterol efflux in the absence and presence of cAMP stimulation similar to full-length apoA-I (Fig. 2A). Cholesterol efflux was dependent on the concentration of the 1/9 chimera reaching maximal levels at 50 µg/ml (Fig. 2B). Helix 9 alone (11-mer peptide) failed to stimulate ABCA1-dependent cholesterol efflux (Fig. 2C) similar to a peptide based on helix 1 (Fig. 1A). Exposure of J774 macrophages concurrently to both helix 9 and helix 1 (not covalently linked) did not stimulate cholesterol efflux, indicating that the two helices needed to be joined to mediate cholesterol efflux via ABCA1 (data not shown).
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-helix is not required for mediating cholesterol efflux via ABCA1.
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32 Å down the length of the joined helices (arrows in Fig. 4B). These similarities on the polar surface in addition to high lipid binding affinity (Table I) may explain why the 1/9 helical peptide mediated ABCA1-dependent cholesterol efflux in a manner not unlike that of the 9/10 helical peptide. Specificity of Cholesterol Efflux for the Structure Created by the 1/9 ChimeraTo evaluate whether the cholesterol efflux capability of the 1/9 chimera was dependent specifically on the presence of helix 9, this 11-mer segment was replaced with helix 3, which represents the other 11-mer helix present within the C-terminal domain of apoA-I. The resulting 1/3 chimera failed to mediate cellular cholesterol efflux in an ABCA1-dependent manner, indicating that helix 9 was unique and critical to the cholesterol efflux properties of the 1/9 peptide (Fig. 5A). The 1/3 chimera also poorly solubilized a turbid solution of DMPC (Fig. 5B and Table I). Edmundson helical wheel projections of the 1/3 chimera revealed that this combination exhibited a narrow hydrophobic surface as polar residues were dispersed around most of the structure (Fig. 5C). This is in keeping with the low amphiphilicity (i.e. hydrophobic moment) of the 1/3 chimera as shown in Table I. These results indicate that a 33-mer chimera with relatively low lipid binding affinity and poor amphipathic character is not able to mediate cellular cholesterol efflux in an ABCA1-dependent manner.
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Cholesterol Efflux Capability of 10/9 and 9/1 Helix Transposition PeptidesTo gain additional insights into the structural determinants that are important for mediating cholesterol efflux via ABCA1, we transposed helices 9 and 10 to create a 10/9 synthetic peptide. This transposition strategy introduces a positively charged residue (Lys-238) into the alignment of negatively charged amino acids formed along the length of the joined 10 plus 9 helical segments, analogous to the structure created by the 4/9 chimera (Fig. 5D). The 10/9 transposition peptide failed to stimulate ABCA1-dependent cholesterol efflux (Fig. 6, A and B) despite the fact that the peptide exhibited class Y structure as well as the same hydrophobicity and amphiphilicity as the native 9/10 helical combination (Table I). The 10/9 peptide effectively cleared a turbid solution of DMPC as indicated in Table I. In contrast, a transposition peptide consisting of helices 9 and 1 (9/1 peptide) stimulated cholesterol efflux in an ABCA1-dependent manner (Fig. 6, A and B). This is consistent with the structure shown in Fig. 6D whereupon transposing helices 1 and 9 created a new alignment of negatively charged residues (Glu-62, Asp-48, Asp-51, and Asp-213) not interrupted by positively charged residues. The alignment of negatively charged amino acids within the 9/1 transposition peptide includes residue Asp-51, positioned 360 degrees and 5 helical turns from Pro-209 (Fig. 6, C and D). These observations support the premise that the topography of negatively charged residues on the polar surface of a 33-mer helical peptide is an important determinant endowing the peptide with cholesterol efflux activity.
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| DISCUSSION |
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-helices. In this regard, a linear array of negatively charged amino acids spanning 32 Å down the long axis of the 9/10 helical element was found to be important for mediating cholesterol efflux and stabilizing ABCA1.
Recent studies indicate that deletion of helix 10 (
220243) and/or helices 9 plus 10 (
209243) from full-length apoA-I greatly diminishes (
80%) ABCA1-dependent cholesterol efflux (23, 24). These published observations are consistent with our findings that the 9/10 helical combination represents a key structure capable of mediating cholesterol efflux via ABCA1. Extensively truncated forms of apoA-I including deletion mutants
165220 (deletion of helices 7, 8, and 9) as well as
141/
185243 (deletion of the N terminus and helices 8, 9 and 10) exhibit normal cholesterol efflux capability (23, 24, 27, 41). There are at least two possible explanations for these findings. First, the apoA-I molecule may possess more than one cholesterol efflux-mediating structural element created by the joining of several complementary amphipathic
-helices. This being the case, apoA-I is likely to possess an additional helical region that plays a role in stabilizing ABCA1 in the plasma membrane. Second, the joining of two non-adjacent helices brought about by the deletion of one or more 22-mer helical segment(s) could conceivably create a structural element similar to the native 9/10 helical combination. Our results with the 1/9 chimera showing stimulation in ABCA1-dependent cholesterol efflux supports this premise, suggesting that various amphipathic
-helices can be combined to create a "prototypic" structural element that is sufficient for mediating cholesterol efflux via ABCA1. Analyses of various combinations of apoA-I
-helices suggest that the joining of helices 6 and 10 (6/10), 7/10, and 8/10 brings together an alignment of acidic residues that may endow cholesterol efflux capability (data not shown). This could explain why various helix-deletion mutants of apoA-I stimulate cholesterol efflux in an ABCA1-dependent manner (23).
It is well known that many of the exchangeable helical apolipoproteins can stimulate cellular cholesterol efflux (4244). Although speculative, the structure created by the 1/9 and 9/10 helical combinations may constitute a motif in the exchangeable apolipoproteins that facilitates the necessary interactions with the ABCA1 transporter to mediate cellular cholesterol efflux. Helix length and lipid binding affinity as well as the topography of negatively charged amino acids on the polar surface of amphipathic
-helices appear to be important determinants governing the cellular cholesterol efflux and ABCA1 stabilization activities of apoA-I. Consideration of each of these determinants may facilitate the identification of a "common" structural element that enables apolipoproteins to mediate ABCA1-dependent cholesterol efflux. The alignment of negatively charged residues spanning the 9/10 helical structure is highly conserved evolutionarily in species such as the mouse, rat, cow, tree shrew, and rainbow trout despite differences in primary amino acid sequences (data not shown). This supports the idea that the topography of acidic residues along the polar surface of the native 9/10 helical region of apoA-I is of biological importance in mediating ABCA1-dependent cholesterol efflux. Moreover, the absence of an obvious binding sequence that is shared among the plasma apolipoproteins does not rule -out the possibility that a specific helical motif is involved in mediating ABCA1-dependent cholesterol efflux. As we have shown, different
-helices with distinct primary amino acid sequences can be combined to create a similar element with a polar surface of aligned negatively charged residues that may be involved in mediating direct protein-protein interactions with ABCA1.
In contrast to the topography of negatively charged residues, which is highly conserved evolutionarily, the class Y structure of the 9/10 segment is not conserved, as exemplified in the mouse, where the region exhibits class A character (data not shown). This suggests that class Y structure per se is not required to stimulate ABCA1-dependent cholesterol efflux, consistent with our studies utilizing the 1/9 chimera. However, replacement of Lys-238 (which confers class Y structure) with a negatively charged residue reduces the ability of apoA-I to stimulate ABCA1-dependent cholesterol efflux (23). This effect was attributed to a decrease in lipid binding affinity of the K238E variant compared with wild-type apoA-I, as judged using a DMPC clearance assay (23). The K238E substitution alters the net charge of the 9/10 segment by 2, which could have contributed to the decrease in lipid binding affinity.
Studies employing peptide 37pA suggest that helical apolipoproteins do not interact with ABCA1 via a traditional receptor/ligand-type mechanism (45, 46). This is based on the findings that peptide 37pA composed of unnatural D-amino acids stimulates cholesterol efflux and stabilizes ABCA1. D-Amino acids are the stereoisomers of L-amino acids that form left-handed helical structures instead of the naturally occurring right-handed helices. Thus, these previous studies provide evidence that there is no stereoselective requirement for helical peptides to mediate cellular lipid efflux via ABCA1 (45). However, as discussed by Remaley et al. (45), studies employing 37pA do not rule out the possibility that protein-protein interactions between helical apolipoproteins and ABCA1 are important for cholesterol efflux. Peptide 37pA is composed of two 18-mer helices (18A) joined via a proline residue. Helical wheel projections and en face cylindrical diagrams of 18A indicate that the structure possesses an alignment of negatively charged residues that is "kinked," spanning
5 helical turns, that is somewhat similar to the 1/9 chimera (data not shown). The topography of negatively charged residues on the polar surface of 18A is not likely to be altered by the use of D-amino acids. Therefore, the idea that protein-protein interactions between helical apolipoproteins and ABCA1 are required for cholesterol efflux and ABCA1 stabilization remains a possibility. Peptide 37pA appears to be potent mediator of cellular cholesterol efflux (45, 46). This suggests that the extremely high lipid binding affinity of the helical peptide may compensate for a less than perfect topography of negatively charged residues on its polar surface by facilitating cholesterol efflux via ABCA1-dependent and -independent mechanisms (45).
Our present findings together with previously published studies support the basic concept that lipid affinity is important for helical apolipoproteins to interact with ABCA1-expressing cells, perhaps by facilitating the binding to lipid micro-domains created by active ABCA1. The high lipid-binding affinity of specific helical segments is also likely to play an important role in stabilizing the structure of nascent HDL. However, we propose that the polar surface of the 9/10 helical element may be required for apoA-I to form a molecular complex with ABCA1 via electrostatic attraction and salt-bridge formation. The present study provides evidence that the same set of apoA-I structural determinants that imparts cholesterol efflux capability is also able to prevent cellular degradation of the ABCA1 transporter. Indeed, the 1/9 chimera and 9/10 helical peptide appear to possess all the necessary structural determinants required for stimulating cholesterol efflux and stabilizing ABCA1. This greatly simplifies future structure/function studies because a finite set of potential variables exist within the context of the 9/10 helical peptide that can be manipulated to precisely define the molecular basis for the interaction between helical apolipoproteins and the ABCA1 transporter. As such, structural analogs of the 9/10 helical peptide may prove useful for identifying what is required for helical apolipoproteins to mediate cholesterol efflux via ABCA1. The observation that a single structural element possesses dual functionality as a stabilizer of ABCA1 and mediator of cellular cholesterol efflux has clinical value for devising small molecule therapeutics for reversing the macrophage foam-cell phenotype and combating atherosclerosis.
| FOOTNOTES |
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|| To whom correspondence should be addressed: Lawrence Berkeley National Laboratory, Donner Laboratory, MS1-224, University of California at Berkeley, One Cyclotron Rd., Berkeley, CA 94720. Tel.: 510-495-2208; E-mail: Jkbielicki{at}lbl.gov.
1 The abbreviations used are: HDL, high density lipoprotein; CPT-cAMP, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate; ABCA1, ATP binding cassette transporter A1; aa, amino acids; DMPC, dimyristoylphosphatidylcholine. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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