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• W2132804909 abstract "It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated “high-capacity binding site” (HCBS). Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Δ187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis. It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated “high-capacity binding site” (HCBS). Glyburide drastically reduced 125I-apoA-I binding to the HCBS, whereas 125I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Δ187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis. apolipoprotein A-I 9-cis-retinoic acid two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis dithiobis(succinimidylpropionate) high-capacity binding site intracellular compartment nascent apolipoprotein A-I-containing particle reconstituted high density lipoprotein particle, 22-(R)-hydroxycholesterol phosphatidylcholine-specific phospholipase C plasma membrane sphingomyelinase wild-type A growing body of evidence indicates that both apolipoprotein A-I (apoA-I) and ABCA1 not only play a major role in HDL biogenesis and in the reverse cholesterol transport process but also have emerged as potential targets for therapies designed to inhibit the development of atherosclerotic vascular disease (1.Brewer Jr., H.B. Remaley A.T. Neufeld E.B. Basso F. Joyce C. Regulation of plasma high-density lipoprotein levels by the ABCA1 transporter and the emerging role of high-density lipoprotein in the treatment of cardiovascular disease.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1755-1760Crossref PubMed Scopus (148) Google Scholar, 2.von Eckardstein A. Hersberger M. Rohrer L. Current understanding of the metabolism and biological actions of HDL.Curr. Opin. Clin. Nutr. Metab. Care. 2005; 8: 147-152Crossref PubMed Scopus (131) Google Scholar). Therefore, there is a strong scientific rationale for a clear understanding of the mechanisms and molecular pathways underlying the apoA-I lipidation process, especially the mechanistic basis of lipid-free apoA-I interaction with ABCA1. ApoA-I binding to the extracellular domain of ABCA1 results in the active removal of cellular cholesterol and phospholipids to lipid-poor apolipoproteins from a variety of cells (3.Brewer Jr., H.B. Santamarina-Fojo S. New insights into the role of the adenosine triphosphate-binding cassette transporters in high-density lipoprotein metabolism and reverse cholesterol transport.Am. J. Cardiol. 2003; 91: 3E-11EAbstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 4.Krimbou L. Marcil M. Genest J. New insights into the biogenesis of human high-density lipoproteins.Curr. Opin. Lipidol. 2006; 17: 258-267Crossref PubMed Scopus (45) Google Scholar). This process plays crucial roles in both the formation and maintenance of HDL levels in plasma and is likely important for the first step of the reverse cholesterol transport process from peripheral tissues, including macrophages in the vessel wall (5.Wang N. Tall A.R. Regulation and mechanisms of ATP-binding cassette transporter A1-mediated cellular cholesterol efflux.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (215) Google Scholar). Although the molecular basis for the interaction between ABCA1 and apoA-I has yet to be elucidated, there are two prevailing hypotheses describing the interaction. First, the direct association model proposes that ABCA1 acts as a receptor to which the apoA-I ligand binds directly. This direct interaction is proposed to stimulate ABCA1's cholesterol efflux activity, resulting in the transfer of cholesterol and phospholipids onto the acceptor apolipoprotein. Evidence for the direct association model comes from chemical cross-linking studies performed in our laboratory and by others, which indicate that apoA-I and ABCA1 are in very close proximity (<7 Å) (6.Chroni A. Liu T. Fitzgerald M.L. Freeman M.W. Zannis V.I. Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1.Biochemistry. 2004; 43: 2126-2139Crossref PubMed Scopus (95) Google Scholar, 7.Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest Jr, J. Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).J. Biol. Chem. 2004; 279: 7384-7394Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 8.Fitzgerald M.L. Morris A.L. Chroni A. Mendez A.J. Zannis V.I. Freeman M.W. ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux.J. Lipid Res. 2004; 45: 287-294Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 9.Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of apoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). A second model has been proposed suggesting that ABCA1 acts by flipping phospholipids to the outer leaflet of the plasma bilayer. Subsequently, apoA-I is proposed to bind these translocated phospholipids and then extract both phospholipid and cholesterol in a process that requires no direct interaction between the apolipoprotein and ABCA1 (10.Chambenoit O. Hamon Y. Marguet D. Rigneault H. Rosseneu M. Chimini G. Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter.J. Biol. Chem. 2001; 276: 9955-9960Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 11.Burgess J.W. Kiss R.S. Zheng H. Zachariah S. Marcel Y.L. Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux.J. Biol. Chem. 2002; 277: 31318-31326Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This view gained support from studies showing that an intact ABCA1 ATPase activity was required for apoA-I binding to the cell surface (10.Chambenoit O. Hamon Y. Marguet D. Rigneault H. Rosseneu M. Chimini G. Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter.J. Biol. Chem. 2001; 276: 9955-9960Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). On the other hand, it has been suggested that the ABCA1-mediated apoA-I lipidation process may occur inside the cell as part of a retroendocytosis pathway (12.Takahashi Y. Smith J.D. Cholesterol efflux to apolipoprotein AI involves endocytosis and resecretion in a calcium-dependent pathway.Proc. Natl. Acad. Sci. USA. 1999; 96: 11358-11363Crossref PubMed Scopus (204) Google Scholar, 13.Neufeld E.B. Stonik J.A. Demosky Jr., S.J. Knapper C.L. Combs C.A. Cooney A. Comly M. Dwyer N. Blanchette-Mackie J. Remaley A.T. et al.The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease.J. Biol. Chem. 2004; 279: 15571-15578Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). These contrasting models demonstrate that the molecular mechanism by which ABCA1 mediates the lipidation of apoA-I has yet to be clarified. In the present study, we investigate the structural characteristics of cellular binding sites for apoA-I and their functions related to the biogenesis of nascent HDL particles. For the present study, we selected fibroblasts from three normal control subjects and one patient with Tangier disease (homozygous for Q597R at the ABCA1 gene), as described previously (14.Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. Dam M.van Yu L. Brewer C. Collins J.A. Molhuizen H.O. et al.Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1481) Google Scholar). The protocol for the study was reviewed and accepted by the Research Ethics Board of the McGill University Health Center. Separate consent forms for blood sampling, DNA isolation, and skin biopsy were provided. Human skin fibroblasts were obtained from 3.0 mm punch biopsies of the forearm of patients and healthy control subjects and were cultured in DMEM supplemented with 0.1% nonessential amino acids, penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS. BHK cells stably transfected with an ABCA1 cDNA that is inducible by treating the cells with mifepristone and cells transfected with the same vectors lacking the ABCA1 cDNA insert (mock-transfected) were generously provided by Dr. John F. Oram from the Department of Medicine, University of Washington, and were characterized and cultured as described previously (15.Oram J.F. Vaughan A.M. Stocker R. ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol.J. Biol. Chem. 2001; 276: 39898-39902Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 16.Vaughan A.M. Oram J.F. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions.J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). These BHK cells do not normally express ABCA1. Purified plasma apoA-I (Biodesign) was resolubilized in 4 M guanidine HCl and dialyzed extensively against PBS. Freshly resolubilized apoA-I was iodinated with 125I by Iodo-Gen® (Pierce) to a specific activity of 3,000–3,500 cpm/ng apoA-I and used within 48 h. Expression of wild-type (WT) apoA-I and its mutants in a bacterial system, and the isolation and purification of these proteins, have been described previously by Marcel and colleagues (17.Frank P.G. Bergeron J. Emmanuel F. Lavigne J.P. Sparks D.L. Denefle P. Rassart E. Marcel Y.L. Deletion of central alpha-helices in human apolipoprotein A-I: effect on phospholipid association.Biochemistry. 1997; 36: 1798-1806Crossref PubMed Scopus (35) Google Scholar). Deletion mutant apoA-I Δ(187-234) was provided by Dr. Yves L. Marcel. Complexes comprising apoA-I and POPC were prepared using the sodium cholate dialysis method as described by Jonas, Steinmetz, and Churgay (18.Jonas A. Steinmetz A. Churgay L. The number of amphipathic alpha-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles.J. Biol. Chem. 1993; 268: 1596-1602Abstract Full Text PDF PubMed Google Scholar). An apoA-I/POPC molar ratio of 1:100 was used in this experiment. Reconstituted HDL particles (rLpA-I) were further concentrated by ultrafiltration (spiral ultrafiltration cartridge, molecular weight cut off 50,000; Amicon) to discard any lipid-free apoA-I or proteolytic peptides. ApoA-I-lipid complex formation was verified by analysis with two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis (2D-PAGGE), as described previously (19.Krimbou L. Marcil M. Davignon J. Genest Jr, J. Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.J. Biol. Chem. 2001; 276: 33241-33248Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Fibroblasts were grown to confluence in 100 mm diameter dishes and then stimulated with 2.5 μg/ml 22-(R)-hydroxycholesterol (22OH) and 10 μM 9-cis-retinoic acid (9CRA) for 20 h in DMEM/BSA. BHK cells stably expressing ABCA1 or mock-transfected cells were treated or not with mifepristone as described by Oram, Vaughan, and Stocker (15.Oram J.F. Vaughan A.M. Stocker R. ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol.J. Biol. Chem. 2001; 276: 39898-39902Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and Vaughan and Oram (16.Vaughan A.M. Oram J.F. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions.J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Cells were incubated in the presence of 10 μg/ml apoA-I for various periods of time or increasing concentrations of apoA-I. Cells were then washed three times with PBS. Dithiobis(succinimidylpropionate) (DSP) was dissolved immediately before use in DMSO. The cross-linking reaction was performed at room temperature in 6 ml/dish cross-linking solution for 30 min using 1 mM DSP, as described by Tall and colleagues (20.Wang N. Silver D.L. Thiele C. Tall A.R. ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein.J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Cells were then washed twice with PBS and lysed at 4°C with immunoprecipitation buffer containing 20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, and 0.5% dodecylmaltoside (Roche) in the presence of a protease inhibitor cocktail (Roche Diagnostics). Inactivation of the cross-linker was done by the addition of Tris (pH 7.5) to a final concentration of 20 mM. Samples containing 125I-apoA-I cross-linked to ABCA1 (200 μg of total protein) were incubated with 10 μl of an affinity-purified human anti-ABCA1 antibody (Novus) for 20 h at 4°C, followed by the addition of Protein A bound to Sepharose (30 μl). The amount of bound iodinated apoA-I to ABCA1 (immunoprecipitates) or non-ABCA1-associated (supernatants) was determined by γ-counting, protein concentration was determined, and results were expressed as ng apoA-I/μg cell protein. However, both the incomplete cross-linking and immunoprecipitation of the apoA-I/ABCA1 complex may result in inaccurate quantification. Several controls were used to verify this important point. Different conditions of cross-linking and immunoprecipitation were used. Furthermore, we found that the cross-linking is effectively completed by 10 min, using 1 mM DSP. Cholesterol efflux was determined as described previously (7.Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest Jr, J. Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).J. Biol. Chem. 2004; 279: 7384-7394Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) with minor modifications. Briefly, 50,000 cells were seeded on 12-well plates. At midconfluence, the cells were labeled with 4–8 μCi/ml [3H]cholesterol (Perkin-Elmer) for 24 h. At confluence, cells were cholesterol-loaded (20 μg/ml) for 24 h. During a 24 h equilibration period, cells were stimulated with 2.5 μg/ml 22OH and 10 μM 9CRA for 20 h. Cholesterol efflux were determined at either 2 h or 4 h with either 10 μg/ml apoA-I-HDL3 or 3 μg/ml apoA-IΔ(187-243). Cellular cholesterol efflux was determined as follows: 3H cpm in medium/(3H cpm in medium + 3H cpm in cells). The results are expressed as percentage of total radiolabeled cholesterol. Confluent fibroblasts were stimulated with 2.5 μg/ml 22OH and 10 μM 9CRA for 20 h. BHK cells stably expressing ABCA1 or mock-transfected cells were treated or not with mifepristone. Cells were incubated in the presence of 10 μg/ml apoA-I for various time periods or for 45 min at 37°C with increasing concentrations of apoA-I. Cells were then washed three times with PBS, and surface proteins were biotinylated with 500 μg/ml sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (Pierce) for 30 min at 4°C. The biotinylation reaction was quenched for 10 min at 4°C by the addition of 1 M Tris-HCl (pH 7.5) to the reaction mixture to a final concentration of 20 mM. Cells were washed twice with ice-cold PBS, lysed, and homogenized, and 200 μg of protein was added to 30 μl of streptavidin-Sepharose (Amersham Biosciences) beads and incubated overnight on a platform mixer at 4°C. The pellet [plasma membrane (PM)] or supernatant [intracellular compartment (ICC)] were washed two times with immunoprecipitation buffer and counted directly for radioactivity. To verify whether the biotinylation of surface protein is complete under the conditions used in the present experiment, the amount of biotin was increased up to 2 mg/ml. No significant additional association of 125I-apoA-I with the PM or the ICC was observed. Confluent fibroblasts were incubated for 45 min in DMEM containing 10 μg/ml 125I-apoA-I. After washing to remove unbound 125I-apoA-I, cells were subjected to treatment with 2.5 U/ml phosphatidylcholine-specific phospholipase C (PC-PLC) or 0.4 U/ml sphingomyelinase (SMase) (Sigma) for 30 min at 37°C. After the washes, the cells were treated for cross-linking or cell surface biotinylation as described above. Cell membrane integrity and cellular toxicity under treatment with phospholipases were assessed by leakage of [3H]adenine (21.Shirhatti V. Krishna G. A simple and sensitive method for monitoring drug-induced cell injury in cultured cells.Anal. Biochem. 1985; 147: 410-418Crossref PubMed Scopus (64) Google Scholar). Cells were separated into Triton X-100-soluble and insoluble fractions as described previously (22.Brown D.A. Rose J.K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2588) Google Scholar). Briefly, confluent 22OH/9CRA-stimulated cells were washed in PBS and then scraped into MES-buffered saline (25 mM MES, pH 6.5, and 0.15 M NaCl) containing 1% Triton X-100 in the presence of a protease inhibitor cocktail. The suspension was homogenized, kept on ice for 20 min, and centrifuged at 14,000 g for 20 min at 4°C. The supernatant (containing the Triton X-100-soluble fraction) was removed, and the pellet was suspended in Triton X-100 buffer containing HEPES (pH 7.4) instead of MES and incubated at room temperature for 30 min to solubilize rafts. The amount of 125I-apoA-I associated with the supernatants or pellets was determined by γ-counting. Aliquots of the supernatant and solubilized pellet were used for immunoblotting and ABCA1 immunoprecipitation. Removal of lipid-free apoA-I from nascent apolipoprotein A-I-containing particles (LpA-I) and separation of lipoproteins by 2D-PAGGE were performed as described previously (19.Krimbou L. Marcil M. Davignon J. Genest Jr, J. Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.J. Biol. Chem. 2001; 276: 33241-33248Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23.Krimbou L. Hajj H.H. Blain S. Rashid S. Denis M. Marcil M. Genest J. Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines.J. Lipid Res. 2005; 46: 1668-1677Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Results were compared statistically by the t-test. Two-tailed P values of <0.05 were considered significantly different. To examine the interaction between apoA-I and ABCA1 in a cell culture model, we first developed a quantitative assay based on optimal chemical cross-linking of 125I-apoA-I to ABCA1 with the cell-permeable DSP followed by immunoprecipitation of the complex with an affinity-purified polyclonal anti-ABCA1, as described in Methods. The amount of bound iodinated apoA-I to ABCA1 (immunoprecipitates) or 125I-apoA-I nonassociated with ABCA1 (supernatants) was determined by γ-counting. As shown in Fig. 1B, 125I-apoA-I (10 μg/ml) was incubated with intact 22OH/9CRA-stimulated fibroblasts for 45 min at 37°C, followed by cross-linking and immunoprecipitation with an anti-ABCA1 antibody. Unexpectedly, the majority of 125I-apoA-I was found nonassociated with ABCA1. We have assumed that the cross-liking reaction was complete based on results obtained with different concentration times of incubation with DSP (see Methods). Therefore, the concentration of 1 mM DSP and the incubation period of 30 min as well as 10 μl of ABCA1 antibody for the immunoprecipitation were used throughout the present study. Importantly, a 45 min period of incubation of apoA-I with cells was chosen to permit sufficient time for the equilibration of apoA-I with different cellular compartments without interfering with the completion of the cross-linking reaction. It is possible, however, that the majority of 125I-apoA-I found nonassociated with ABCA1 reflects the inefficiency of the cross-linking procedure. Several controls were used to verify this important point. Transferrin, a ligand that is known to specifically interact with the transferrin receptor, was used to verify the efficiency of the cross-linking reaction. 125I-transferrin was incubated with HepG2 for 45 min at 37°C, followed by cross-linking with DSP (1 mM, 30 min) and immunoprecipitation of the cross-linked 125I-transferrin/transferrin receptor complex using polyclonal anti-transferrin receptor antibody. We found that 92% of 125I-transferrin coimmunoprecipitated with the transferrin receptor antibody (data not shown). This result supports the efficiency of the cross-linking reaction under similar conditions used for the cross-linking of apoA-I/ABCA1 described above. The apoA-I/ABCA1 cross-linking assay permitted separation between the ABCA1 binding site and other eventual potential cellular sites for the binding of apoA-I, such as scavenger receptor class B type I, ABCG1, or specialized phospholipid membrane domains. However, there is no detectable presence of scavenger receptor class B type I or ABCG1 in human fibroblasts, as reported previously (24.O'Connell B.J. Denis M. Genest J. Cellular physiology of cholesterol efflux in vascular endothelial cells.Circulation. 2004; 110: 2881-2888Crossref PubMed Scopus (81) Google Scholar). To test the specificity of the cross-liking and immunoprecipitation methods, the presence of a 30-fold excess of unlabeled apoA-I during the incubation of 125I-apoA-I with 22OH/9CRA-stimulated fibroblasts, the absence of 22OH/9CRA treatment, or ABCA1 mutant fibroblasts (Q597R) drastically reduced the larger fraction of 125I-apoA-I nonassociated with ABCA1 (Fig. 1B). Furthermore, the minor fraction of 125I-apoA-I associated with ABCA1 was also reduced. This result is consistent with the total binding of 125I-apoA-I to the cell under different conditions (Fig. 1A). The observation that the presence of ABCA1 mutant Q597R abolished the association of 125I-apoA-I to both ABCA1 associated and nonassociated fractions indicates that a functional ABCA1 is required for the formation of the larger fraction of 125I-apoA-I nonassociated with ABCA1. To verify that the immunoprecipitates contained the majority of ABCA1, the immunoprecipitates and supernatants of 22OH/9CRA-stimulated fibroblasts in the presence and absence of DSP were analyzed by 4–22.5% SDS-PAGE under nonreducing conditions. 125I-apoA-I was detected by direct autoradiography, and ABCA1 was revealed by an anti-ABCA1 antibody. As shown in Fig. 1C (upper panel), in the presence of DSP, the majority of 125I-apoA-I was found in the supernatants as monomeric and multimeric forms, whereas a minor proportion of 125I-apoA-I was found associated with ABCA1 in the immunoprecipitates. At the same time, no detectable ABCA1 was found in the supernatants, and the majority of the oligomeric ABCA1 complex was found in the immunoprecipitates, including the tetramers, as reported previously (25.Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest J. Characterization of oligomeric human ATP binding cassette transporter A1. Potential implications for determining the structure of nascent high density lipoprotein particles.J. Biol. Chem. 2004; 279: 41529-41536Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 26.Trompier D. Alibert M. Davanture S. Hamon Y. Pierres M. Chimini G. Transition from dimers to higher oligomeric forms occurs during the ATPase cycle of the ABCA1 transporter.J. Biol. Chem. 2006; 281: 20283-20290Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 125I-apoA-I treated with DSP in the absence of the cell was used as a standard. On the other hand, 125I-apoA-I associated or not with ABCA1 was found in its monomeric form in the absence of DSP (Fig. 1C, lower panel). This result is consistent with the quantitative analysis reported in Fig. 1B. The fact that the majority of ABCA1 was immunoprecipitated with the anti-ABCA1 antibody in the absence or presence of DSP indicates that the cross-linking with DSP did not affect the interaction of ABCA1 with the antibody. Together, these results support the specificity and the efficiency of both the cross-linking and immunoprecipitation methods and validate our quantitative assay. Based on the experiments described above, we initially hypothesized that apoA-I nonassociated with ABCA1 may represent a new cellular apoA-I binding site. To further examine the role of ABCA1 in mediating the formation of this new, non-ABCA1 apoA-I binding site, different levels of ABCA1 were overexpressed in BHK cells with a mifepristone-inducible ABCA1 gene (15.Oram J.F. Vaughan A.M. Stocker R. ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol.J. Biol. Chem. 2001; 276: 39898-39902Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 16.Vaughan A.M. Oram J.F. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions.J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). As shown in Fig. 2A (inset), in the absence of induction, BHK-ABCA1 cells had no detectable ABCA1 protein, whereas cells induced with 0.1 or 10 nM mifepristone for 20 h expressed increasing levels of ABCA1 in a dose-dependent manner. Concomitantly, cellular cholesterol efflux was increased significantly (2 ± 0.35, 8 ± 0.22, and 13 ± 0.68% at 0, 0.1, and 10 nM mifepristone, respectively). At the same time, the total binding of 125I-apoA-I was also increased significantly by increasing the dose of mifepristone (Fig. 2A). On the other hand, the putative non-ABCA1 apoA-I binding site was increased significantly after induction with 10 nM mefipristone compared with 0.1 nM (Fig. 2B). Similarly, the smaller fraction of apoA-I associated with ABCA1 was also increased. These results support the data obtained with 22OH/9CRA-stimulated fibroblasts and indicate that ABCA1 is involved in the formation of the new putative non-ABCA1 apoA-I binding site. To better characterize the new binding site for apoA-I, intact normal fibroblasts treated or not with 22OH/9CRA or ABCA1 mutant Q597R were incubated with increasing concentrations of 125I-apoA-I for 45 min at 37°C. After washing to remove unbound 125I-apoA-I, cross-linking, immunoprecipitation, and quantification of 125I-apoA-I associated or not with ABCA1 were performed as described above. As shown in Fig. 3A, 125I-apoA-I exhibited saturable binding to both ABCA1 and non-ABCA1 binding sites that was found to occur in a concentration-dependent manner in 22OH/9CRA-s" @default.
• W2132804909 created "2016-06-24" @default.
• W2132804909 creator A5001665358 @default.
• W2132804909 creator A5004683846 @default.
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• W2132804909 date "2007-11-01" @default.
• W2132804909 modified "2023-09-26" @default.
• W2132804909 title "Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis" @default.
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