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Originally published In Press as doi:10.1074/jbc.M400561200 on March 29, 2004

J. Biol. Chem., Vol. 279, Issue 23, 24044-24052, June 4, 2004
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Identification of an Apolipoprotein A-I Structural Element That Mediates Cellular Cholesterol Efflux and Stabilizes ATP Binding Cassette Transporter A1*

Pradeep Natarajan{ddagger}, Trudy M. Forte{ddagger}, Berbie Chu{ddagger}, Michael C. Phillips§, John F. Oram¶, and John K. Bielicki{ddagger}||

From the {ddagger}Lawrence Berkeley National Laboratory, Donner Laboratory MS1–224, 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
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44–65) and 10 (amino acids 220–241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209–219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic {alpha}-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 {alpha}-helices is required to mediate cholesterol efflux and stabilize ABCA1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated levels of plasma HDL1 cholesterol are associated with reduced risk of atherosclerosis (1, 2). The beneficial effects of HDL are related in part to its role in reverse cholesterol transport. The first step in the anti-atherogenic reverse cholesterol transport pathway involves the efflux of cholesterol from macrophage foam cells in the artery wall mediated by lipidpoor apolipoprotein (apo) A-I (3, 4). Lipid efflux mediated by apoA-I generates nascent HDL, reverses the macrophage foamcell phenotype, and is clinically relevant in humans for protecting against atherosclerosis (5–7).

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 (8–13). 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 (18–20). 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 44–243) 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 {alpha}-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 {alpha}-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 {alpha}-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 44–65) and 10 (aa 220–241) 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 {alpha}-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 {alpha}-helices derived from apoA-I mediate cholesterol efflux via ABCA1 and/or if multiple {alpha}-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 {alpha}-helices is required to create a specific structural element with both cholesterol efflux and ABCA1 stabilization properties.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic Peptides—Helical peptides used in this study were composed of sequences of amino acids as found in the C-terminal domain (aa 44–243) of apoA-I using the convention of Mishra et al. (25) to define the amphipathic {alpha}-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 44–65 (22-mer); helix 9, aa 209–219 (11-mer); helix 10, aa 220–241 (22-mer); 1/9 chimera, aa 44–65/209–219 (33-mer); 1/3 chimera, aa 44–65/88–98 (33-mer); 2/9 chimera, aa 66–87/209–219 (33-mer); 4/9 chimera, aa 99–120/209–219 (33-mer); the 9/10 peptide, aa 209–241 (33-mer); 10/9 transposition peptide, aa 220–241/209–219 (33-mer), and 9/1, aa 209–219/44–65 (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 {alpha}-helices (28). Stock solutions (0.5–1 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-I—A 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 Protocol—J774 macrophages were used to assess the cholesterol efflux properties of synthetic amphipathic {alpha}-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 Peptides—In 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 Stabilization—J774 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cholesterol Efflux Capability of a Synthetic Peptide Based on Helix 1 of ApoA-I—Helix 1 has high lipid binding affinity; thus, we asked whether a synthetic peptide (22-mer) corresponding to helix 1 of apoA-I promoted cholesterol efflux in an ABCA1-dependent manner using J774 macrophages. The 22-mer helix 1 peptide failed to stimulate ABCA1-dependent cholesterol efflux (Fig. 1A). Cholesterol efflux from cAMP-treated and -untreated cells was equivalent in contrast to the efflux obtained with full-length apoA-I, which increased dramatically upon the up-regulation of the ABCA1 transporter (Fig. 1B). At relatively high concentrations of the helix 1 peptide (i.e. 75 µg/ml), cholesterol efflux was only 15% that obtained with full-length apoA-I using cAMP-treated macrophages (Fig. 1C). These results indicate that the high lipid binding affinity associated with helix 1 was not sufficient to stimulate cholesterol efflux via the ABCA1 transporter.



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  		FIG. 1.
A synthetic peptide (22-mer) based on helix 1 (aa 44–65) of apoA-I fails to mediate cholesterol efflux via ABCA1. Panels and B, J774 macrophages were incubated (12 h) with (circles) and without (squares) a cAMP analog to up-regulate ABCA1 expression. cholesterol efflux properties of the lipid-free form of helix 1 are shown in panel A, and efflux to lipid-free full-length apoA-I is shown in panel B The concentration of each acceptor was 75 µg/ml. Panel C, the dependence of cholesterol efflux on the concentration of the helix 1 peptide; shown are the results using cAMP-treated cells. A-I corresponds to full-length apoA-I (25 µg/ml). Values are the mean ± S.D., n = 3 (separate experiments). Error bars are smaller than symbols when not seen.

 
Cholesterol Efflux Properties of a Chimeric Peptide (33-mer) Composed of ApoA-I Helices 1 and 9 —The results presented in Fig. 1 showing that helix 1 was a poor mediator of cholesterol efflux suggests that several amphipathic {alpha}-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 209–219) 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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  		FIG. 2.
A chimeric peptide composed of apoA-I helices 1 and 9 mediates cholesterol efflux via ABCA1. Panel A, J774 macrophages were treated (12 h) with (closed circles and squares) and without (open circles and squares) a cAMP analog to up-regulate ABCA1 expression. Cholesterol efflux mediated by the helix 1/9 chimera is depicted by the open and closed squares, and full-length apoA-I is depicted by the open and closed circles. Panel B, the dependence of cholesterol efflux on the concentration of the lipid-free form of the 1/9 chimera; shown are the results using cAMP-treated cells. AI corresponds to the lipid-free form of full-length apoA-I (75 µg/ml). Panel C, cholesterol efflux to various acceptors including the 1/9 chimera, full-length apoA-I, helix 1 (aa 44–65), and helix 9 (aa 209–219). Each acceptor was used in lipid-free form at a concentration of 75 µg/ml. Values are the means ± S.D., n = 3.

 
The results presented in Fig. 2 strongly imply that the joining of helices 1 and 9 brought together key determinants that enabled the 33-mer peptide to stimulate cellular cholesterol efflux via ABCA1. However, this joining did not alter the lipid binding affinity compared with helix 1 alone (22-mer), as measured using a surface balance technique (summarized in Table I). The lipid binding affinities of helix 1, the 1/9 chimera, and full-length apoA-I were identical (30, 31, and 32 ± 1 dynes/cm, respectively). The 1/9 chimera was slightly more hydrophobic compared with helix 1, but the amphiphilicity (i.e. hydrophobic moment) of the two peptides was nearly the same (Table I). Both the 1/9 chimera and the helix 1 peptide cleared a turbid solution of DMPC in a similar manner (summarized in Table I). Collectively, these results indicate that the ability of the 1/9 chimera to mediate cholesterol efflux in an ABCA1-dependent manner was not the result of an increase in lipid binding affinity created by the joining of the two helical segments.


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  		TABLE I
Biophysical properties of amphipathic helical peptides derived from human apoA-I

 
Cholesterol Efflux Properties of Helix 10 and the 9/10 Helical Combination—Helix 10 (aa 220–241) is the most hydrophobic helical segment of apoA-I (Table I), but a synthetic 22-mer peptide based on helix 10 was a poor mediator of cellular cholesterol efflux when used at a high concentration of 100 µg/ml (Fig. 3A). In contrast, a 33-mer peptide composed of apoA-I helices 9 and 10 stimulated cholesterol efflux in an ABCA1-dependent manner (Fig. 3B). Maximal levels of cholesterol efflux from cAMP-treated cells were observed at 25 µg/ml 9/10 helical peptide, suggesting that this 33-mer peptide is more efficient than the 1/9 chimera (Fig. 2B versus 3C). ApoA-I stimulated cholesterol efflux to maximal levels at a concentration of 10 µg/ml (data not shown), indicating that the 9/10 peptide was less efficient than the full-length apoA-I. Helix 10 and the 9/10 peptide cleared a turbid solution of DMPC in a similar manner, consistent with their predicted hydrophobicity and hydrophobic moments, which did not differ between the two peptides (Table I). The monolayer exclusion pressure was slightly higher for the 9/10 helical peptide compared with helix 10 alone and the 1/9 chimera (Table I). This may account for the observation that the 9/10 helical peptide was 2-fold more potent than the 1/9 chimera in mediating ABCA1-dependent cholesterol efflux (Figs. 2B versus 3C).



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  		FIG. 3.
A 33-mer peptide composed of helices 9 and 10 of apoA-I mediates cholesterol efflux via ABCA1. Panels A and B, J774 macrophages were incubated with (circles) and without (squares) a cAMP analog as described in Fig. 1 and 2. Panel A, cholesterol efflux mediated by a 22-mer peptide based on helix 10 (aa 220–241) of apoA-I used in lipid-free form at a concentration of 100 µg/ml. Results are representative of at least two independent experiments performed in triplicate. Panel B, the ability of a 33-mer (100 µg/ml) composed of helices 9 and 10 to stimulate cholesterol efflux. Panel C, dependence of cholesterol efflux on the concentration of the 9/10 helical peptide. Shown are the results using cAMP-treated J774 cells. Values shown are the means ± S.D., n = 3.

 
Structure of the 1/9 Chimera and 9/10 Helical Peptide—Fig. 4 illustrates the similarities and differences in the amino acid sequence and structures of the 1/9 and 9/10 helical combinations. Each of the structures consists of 33 amino acids, but the arrangement of the 11- and 22-mer helical segments differ between the 1/9 and 9/10 helical peptides (Fig. 4). Both helical peptides possess amphipathic character with positively charged residues located at the lipid-water interface of the helical structures (Fig. 4A). The 9/10 peptide exhibits class Y structure attributed to Lys-238 at the apex of the helical wheel projection; the 1/9 structure, on the other hand, more closely resembles a class A helix in which the positively charged residues are positioned toward the lipid-water interface. The latter suggests that class Y structure attributed to the specific location of a positively charged residue in the middle of the polar surface of an amphipathic {alpha}-helix is not required for mediating cholesterol efflux via ABCA1.



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  		FIG. 4.
Structural similarities between the 9/10 helical peptide and the 1/9 chimera. Panel A, Edmundson helical wheel projections showing the 9/10 peptide and 1/9 chimera. Shaded circles represent negatively charged residues, and partially shaded circles positively charged amino acids. Dashed lines mark the lipid-water interface of the {alpha}-helices. Panel B, {alpha}-helices are shown as cylinders cut down the long axis of the polar face and flattened. Arrows in all panels show the position of negatively charged residues that form an alignment spanning 32 Å down the length (5–6 turns) of the joined segments.

 
Despite the difference in the distribution of positively charged residues, the net charge of the 1/9 chimera is the same as the 9/10 helical combination (Table I). In addition, the position of negatively charged residues down the length of the joined helical segments is nearly identical for the structures created by the 1/9 and 9/10 helical combinations, as noted in the cylindrical diagrams (Fig. 4B). Three of these negatively charged residues form an alignment spanning ~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 Chimera—To 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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  		FIG. 5.
Cholesterol efflux properties, DMPC clearance, and structures of various chimeric peptides derived from apoA-I amphipathic {alpha}-helices. Panel A, cholesterol efflux experiments using J774 macrophages incubated with (dark bars) and without (open bars) a cAMP analog as described in Fig. 1 and 2. Panel B, DMPC clearance assays with the chimeras; control indicates no peptides added. Results are representative of three experiments. Panel C, Edmundson helical wheel projections showing the structure of the various chimeras. The dashed line corresponds to the lipid-water interface of the amphipathic {alpha}-helices. Panel D, amphipathic {alpha}-helical peptides, shown as cylinders cut down the long axis of the polar face and flattened. Shaded circles correspond to negatively charged amino acids, and partially shaded circles correspond to positively charged residues.

 
Similar helix replacement experiments were conducted utilizing various 22-mer repeats in place of helix 1. A 33-mer chimera composed of helices 2 and 9 (2/9) failed to stimulate ABCA1-dependent cholesterol efflux (Fig. 5A) even though the chimera possessed an alignment of negatively charged residues on its polar surface similar to 1/9 and 9/10 helical peptides (Fig. 5D). The 2/9 chimera possessed amphipathic character (Fig. 5C), but the net charge of the peptide was –5, and the peptide poorly solubilized DMPC (Fig. 5B), consistent with its calculated hydrophobicity (Table I). These results support the premise that relatively good lipid-binding affinity is an important factor for mediating cholesterol efflux via ABCA1. A 33-mer chimera composed of helices 4 and 9 (4/9) also failed to mediate ABCA1-dependent cholesterol efflux (Fig. 5A). However, the 4/9 chimera was found to possess relatively good lipid binding affinity as judged by a DMPC clearance assay (Fig. 5B). The polar surface of the 4/9 chimera was found to be somewhat different compared with that of the 1/9 and 9/10 helical peptides, with positively charged amino acids inserted between negatively charged residues that span the length of the 4/9 helical peptide. These findings support the premise that lipid binding affinity alone is not sufficient to stimulate cholesterol efflux. Factors in addition to lipid binding affinity (i.e. DMPC clearance capability) appear also to be important for a 33-mer helical peptide to mediate cholesterol efflux via ABCA1.

Cholesterol Efflux Capability of 10/9 and 9/1 Helix Transposition Peptides—To 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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  		FIG. 6.
Cholesterol efflux properties of 10/9 and 9/1 transposition peptides. Panel A, J774 macrophages were incubated with (dark bars) and without (open bars) a cAMP analog to up-regulate ABCA1 protein. The ability of transposition peptides 10/9 and 9/1 to stimulate cholesterol efflux is shown. Each peptide was used in lipid-free form at a concentration of 50 µg/ml. Panel B, dependence of cholesterol efflux on the concentration of 10/9 and 9/1 helical peptides. Results are representative of two identical experiments; shown are the results from cAMP-treated cells. Panel C, Edmundson helical wheel projections showing the amphipathic structure of the 10/9 and 9/1 peptides. Panel D, cylindrical diagrams showing the relative positions of amino acids along the {alpha}-helices. Shaded circles highlight the negatively charged residues, and partially shaded circles highlight the positively charged amino acids. The 9/1 peptide was engineered with a proline in place of Leu-44, in keeping with the other 33-mers used in this study.

 
The 1/9 Chimera and 9/10 Helical Peptide Stabilize the ABCA1 Transporter—To assess ABCA1 stabilization, J774 macrophages were treated with a cAMP analog to up-regulate ABCA1 protein expression and then incubated for 6 h without cAMP. In the absence of inducer, ABCA1 protein is rapidly degraded in these cells (Fig. 7). Peptides based on individual 11- and 22-mer helical segments including helices 1, 9, and 10 failed to prevent ABCA1 degradation, providing evidence that high lipid binding affinity alone is not sufficient to stabilize the ABCA1 transporter (Fig. 7). In contrast, at 20 µg/ml the 1/9 chimera and the 9/10 helical peptides stabilized cellular ABCA1 protein to levels comparable with those observed when cells were exposed continuously to cAMP (Fig. 7). Detailed concentration dependence studies revealed that the 1/9 and 9/10 helical peptides prevented ABCA1 degradation at concentrations as low as 10 µg/ml, similar to full-length apoA-I (data not shown). In keeping with the results of the cholesterol efflux studies, the 10/9 transposition peptide failed to prevent ABCA1 degradation, whereas the 9/1 transposition retained ABCA1 stabilization activity (Fig. 7). Thus, it appears that the helical combinations that stimulate cholesterol efflux also stabilize the ABCA1 transporter.



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  		FIG. 7.
Peptides 1/9 and 9/10 stabilize cellular ABCA1 protein. J774 macrophages were incubated (18 h) with 0.5 mM 8-bromo-cAMP in medium containing 0.1% bovine serum albumin to up-regulate ABCA1 protein expression. Washed cells were subsequently exposed for 6 h to bovine serum albumin medium with (+) or without (–) 8-bromo-cAMP and the indicated synthetic peptides (20 µg/ml). None refers to no peptides. The cellular membrane content of ABCA1 protein was measured by immunoblot analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There is currently a controversy as to how apoA-I interacts with ABCA1 to mediate cellular cholesterol efflux and nascent HDL assembly (36–40). This controversy stems in part from the lack of basic information regarding the helical segments and structural determinants of apoA-I that are responsible for stimulating cholesterol efflux via the ABCA1 transporter. We have found in the present study that individual 22-mer helices of apoA-I with high lipid binding affinity, such as helices 1 and 10, neither stimulate ABCA1-dependent cholesterol efflux nor stabilize cellular ABCA1 protein. These observations indicate that factors in addition to lipid binding affinity are required for apoA-I to stimulate cholesterol efflux via ABCA1. The conjoining of apoA-I helices 1 and 9 (1/9 chimera) or helices 9 and 10 (9/10 peptide) endowed cholesterol efflux capability, apparently via ABCA1, as judged utilizing J774 macrophages treated with and without a cAMP analog. As such, the native 9/10 helical combination most likely represents the minimum structure within apoA-I that is required for stimulating cholesterol efflux and stabilizing ABCA1. This suggests that helix length may be an important factor governing the cholesterol efflux capability of apoA-I amphipathic {alpha}-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 ({Delta}220–243) and/or helices 9 plus 10 ({Delta}209–243) 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 {Delta}165–220 (deletion of helices 7, 8, and 9) as well as {Delta}1–41/{Delta}185–243 (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 {alpha}-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 {alpha}-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 {alpha}-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 (42–44). 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 {alpha}-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 {alpha}-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
 
* This work was supported by National Institutes of Health Grants HL059483 (to J. K. B.), HL55493 (to T. M. F.), HL18645 (to J. F. O.), HL55362 (to J. F. O.), and HL22633 (to M. C. P.). The research was conducted at Ernest Orlando Lawrence Berkeley National Laboratory through the United States Department of Energy under Contract DE-AC03-7600098. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| 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. Back


   ACKNOWLEDGMENTS
 
We thank Stephi Berger for helpful comments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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