Efflux of Cellular Cholesterol and Phospholipid to Apolipoprotein A-I Mutants (1996)

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(−6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(−6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(−6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(−6-222)) and 210 (apoA-I-(−6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(−6-150)) and 135 (apoA-I-(−6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(−6-243), apoA-I-(−6-222), and apoA-I-(−6-210), but slightly higher for apoA-I-(−6-150) and apoA-I-(−6-135). When complexed with phospholipid, all forms except apoA-I-(−6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent. Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(−6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(−6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(−6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(−6-222)) and 210 (apoA-I-(−6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(−6-150)) and 135 (apoA-I-(−6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(−6-243), apoA-I-(−6-222), and apoA-I-(−6-210), but slightly higher for apoA-I-(−6-150) and apoA-I-(−6-135). When complexed with phospholipid, all forms except apoA-I-(−6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent. INTRODUCTIONIt is currently accepted that apolipoprotein A-I (apoA-I) 1The abbreviations used are: apoA-Iapolipoprotein A-IERendoplasmic reticulumrHDLreconstituted high density lipoproteinPCphosphatidylcholinePMplasma membranePOPCpalmitoyloleoyl phosphatidylcholine. -containing lipoproteins play a key role in the efflux of cellular cholesterol, the first step in the reverse cholesterol transport pathway (1Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 2Forte T.M. McCall M.R. Curr. Opin. Lipidol. 1994; 5: 354-364Crossref PubMed Scopus (80) Google Scholar, 3Barter P. Curr. Opin. Lipidol. 1993; 4: 210-217Crossref Scopus (62) Google Scholar). Thus, apoA-I-containing lipoproteins are the most efficient initial acceptors of cellular cholesterol (1Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 2Forte T.M. McCall M.R. Curr. Opin. Lipidol. 1994; 5: 354-364Crossref PubMed Scopus (80) Google Scholar), and removal of these lipoproteins from the plasma greatly reduces the release of cholesterol from cells (4Fielding C.J. Moser K. J. Biol. Chem. 1982; 257: 10955-10960Abstract Full Text PDF PubMed Google Scholar). The mechanism of how apoA-I executes this function remains, however, unresolved. It has been demonstrated that short peptides mimicking the secondary structure of apoA-I are able, to a certain extent, to imitate its ability to facilitate removal of cellular cholesterol (5Davidson W.S. Lund-Katz S. Johnson W.J. Anantharamaiah G.M. Palgunachari M.N. Segrest J.P. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1994; 269: 22975-22982Abstract Full Text PDF PubMed Google Scholar, 6Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. J. Clin. Invest. 1994; 94: 1698-1705Crossref PubMed Scopus (175) Google Scholar, 7Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar), suggesting that a rather nonspecific mechanism, which relies mainly on the ability of apoA-I to bind lipids, appears to be involved. On the other hand, new evidence suggests that specific sequences of apoA-I are responsible for cholesterol efflux (8Banka C.L. Black A.S. Curtiss L.K. J. Biol. Chem. 1994; 269: 10288-10297Abstract Full Text PDF PubMed Google Scholar, 9Fielding P.E. Kawano M. Catapano A.L. Zoppo A. Marcovina S. Fielding C.J. Biochemistry. 1994; 33: 6981-6985Crossref PubMed Scopus (85) Google Scholar, 10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar), pointing to the involvement of a more specific and possibly receptor-dependent mechanism.In our previous work using monoclonal antibodies against specific epitopes of apoA-I, we identified a sequence, apoA-I-(140-150), that was associated with the ability of apoA-I to promote efflux of intracellular cholesterol (10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar). In order to further investigate the involvement of various domains of apoA-I in cholesterol efflux, we used recombinant proapoA-I, either intact or truncated, in the lipid-free form or incorporated into reconstituted high density lipoprotein (rHDL), to study the effect of the carboxyl-terminal domain truncations on the efflux of cholesterol and phospholipid from cultured cells. While the proform of apoA-I is a minor component of circulating apoA-I, its known properties do not deviate strongly from mature apoA-I (11Sorci-Thomas M.G. Parks J.S. Kearns M.W. Pate G.N. Zhang C. Thomas M.J. J. Lipid Res. 1996; 37: 673-683Abstract Full Text PDF PubMed Google Scholar, 12Roghani A. Zannis V.I. Biochemistry. 1988; 27: 7428-7435Crossref PubMed Scopus (15) Google Scholar, 13Westman J. Roobol C. Heymans C. Carlson L.A. Wulfert E. Scand. J. Clin. Lab. Invest. 1993; 53: 773-782Crossref PubMed Scopus (12) Google Scholar, 14McGuire K.A. Davidson W.S. Jonas A. J. Lipid Res. 1996; 37: 1519-1528Abstract Full Text PDF PubMed Google Scholar) and the biological principles underlying structure-function relationships between cholesterol efflux and proteins should be equally relevant for mature apoA-I and proapoA-I.DISCUSSIONThe present study continues our investigations into the identification of putative structural features of apolipoprotein A-I that determine its ability to promote efflux of cholesterol and phospholipid from cultured cells. In our previous work we demonstrated that monoclonal antibodies reacting with epitope within the region apoA-I-(140-150) specifically inhibited the ability of apoA-I-containing lipoproteins to promote efflux of intracellular but not plasma membrane cholesterol (10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar). For this study we have produced several versions of recombinant proapoA-I selectively truncated at loci suspected of involvement in cellular cholesterol efflux. The ability of these mutants, either in the lipid-free form or incorporated into rHDL, to mobilize cellular cholesterol and phospholipid were compared.At least two stages have been identified in the process of cholesterol efflux: transfer of cholesterol from intracellular compartments to the plasma membrane and transfer from plasma membrane to the extracellular acceptor (1Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). While the first stage may be receptor-dependent (35Mendez A.J. Oram J.F. Bierman E.L. J. Biol. Chem. 1991; 266: 10104-10111Abstract Full Text PDF PubMed Google Scholar), the second stage mainly depends on the secondary structure of the apolipoproteins (36Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 66-141Abstract Full Text PDF Google Scholar). In the present study, we found that varying degrees of truncation produced marked differences in the ability of lipid-free apoA-I to promote efflux of cellular cholesterol. In contrast to the selective inhibition of the efflux of intracellular cholesterol by monoclonal antibodies (10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar), truncations of apoA-I exerted a similar effect on the efflux of both intracellular and plasma membrane cholesterol. The changes in cholesterol efflux resulting from truncation of apoA-I did not correlate with binding of apoA-I to cells. Taken together, these observations suggest that the removal of the varying proportion of the carboxyl terminus of apoA-I does not affect intracellular stage of cholesterol efflux, a stage that may depend on a specific interaction between apoA-I and cells. Instead, it appears to reflect changes in the ability of truncated forms of apoA-I to interact and form complexes with cholesterol and phospholipid, processes that are associated with the second stage of the efflux.It was suggested by Forte et al. (30Forte T.M. Goth-Goldstein R. Nordhausen R.W. McCall M.R. J. Lipid Res. 1993; 34: 317-324Abstract Full Text PDF PubMed Google Scholar, 31Forte T.M. Bielicki J.K. Goth-Goldstein R. Selmek J. McCall M.R. J. Lipid Res. 1995; 36: 148-157Abstract Full Text PDF PubMed Google Scholar) that association with phospholipid is a prerequisite for the ability of lipid-free apoA-I to facilitate cholesterol efflux. Several findings in this work support that hypothesis. First, the effect of truncations on cholesterol efflux paralleled their effect on the efflux of phospholipid. Second, differences in the ability of the various forms of apoA-I to promote cholesterol efflux disappeared when apoA-I was complexed with phospholipid prior to incubation with cells. The cholesterol/phospholipid ratio in the medium rose quickly during the first 1-3 h of incubation and remained constant thereafter. Taken together, these data suggest that any differences observed in the promotion of cholesterol efflux from HepG2 cells most likely reflect a difference in capacity to associate with cellular phospholipid and subsequently form the apoA-I/phospholipid complexes required for efflux of cholesterol.The data obtained with truncations that involved the last 21 amino acids suggest that this domain of apoA-I plays a crucial role in interaction with cellular lipids. This region includes a class Y amphipathic helix, which is suggested to bind phospholipid strongly and may play an important role in assembling apoA-I into lipid-containing complexes (37Brouillette C.G. Anantharamaiah G.M. Biochim. Biophys. Acta. 1995; 1256: 103-129Crossref PubMed Scopus (166) Google Scholar). It was also demonstrated that truncation of apoA-I to residue 189 (38Holvoet P. Zhao Z. Vanloo B. Vos R. Deridder E. Dhoest A. Taveirne J. Brouwers P. Demarsin E. Engelborghs Y. Rosseneu M. Collen D. Brasseur R. Biochemistry. 1995; 34: 13334-13342Crossref PubMed Scopus (87) Google Scholar), 192 (39Ji Y. Jonas A. J. Biol. Chem. 1995; 270: 11290-11297Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), or 212 (33Minnich A. Collet X. Roghani A. Cladaras C. Hamilton R.L. Fielding C.J. Zannis V.I. J. Biol. Chem. 1992; 267: 16553-16560Abstract Full Text PDF PubMed Google Scholar) significantly diminished its ability to react with phospholipid. Truncation to residues 226, 217, and 201, which decreased the ability of apoA-I to associate with HDL in vivo, also produced a marked increase in the clearance of apoA-I (17Schmidt H.H.-J Remaley A.T. Stonik J.A. Ronan R. Wellmann A. Thomas F. Zech L.A. Brewer H.B. Hoeg J.M. J. Biol. Chem. 1995; 270: 5469-5475Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The important lipid binding properties of this region has been also demonstrated with other techniques; peptide consisting of residues apoA-I-(220-241), together with peptide apoA-I-(44-65), were the only fragments of apoA-I apparently able to bind phospholipid (40Palgunachari M.N. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Alluri S. Anantharamaiah G.M. Segrest J.P. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Crossref PubMed Scopus (202) Google Scholar). In this work we have demonstrated that truncation to residue 222 caused a significant reduction in the ability of apoA-I to promote efflux of cellular lipids suggesting that the 223-243 region is most likely involved in the formation of apoA-I-lipid complex.Unexpectedly, truncation of apoA-I to residue 150 completely restored and even enhanced promotion of cholesterol and phospholipid efflux. At least three explanations can be offered for this finding. First, another cryptic lipid-binding domain exists in the center of the apoA-I, which is unmasked by truncation. The most likely candidates are a “hinge” region apoA-I-(87-142), which contains the only two remaining class Y amphipathic helices of apoA-I and which has a strong ability to bind lipids (37Brouillette C.G. Anantharamaiah G.M. Biochim. Biophys. Acta. 1995; 1256: 103-129Crossref PubMed Scopus (166) Google Scholar), or a region apoA-I-(44-65), which also shows a strong lipid-binding ability (40Palgunachari M.N. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Alluri S. Anantharamaiah G.M. Segrest J.P. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Crossref PubMed Scopus (202) Google Scholar). Second, this other domain is a primary lipid binding domain in full-length apoA-I, but is structurally compromised when the last 21 amino acids are truncated. Third, the lipid-binding properties of apoA-I do not depend on a specific sequence or a domain, but rather on the overall secondary structure of the whole protein which is affected when some parts of the protein are truncated. Evidence from the work with synthetic peptides suggests the existence of two lipid-binding domains in apoA-I and therefore favors the first possibility (40Palgunachari M.N. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Alluri S. Anantharamaiah G.M. Segrest J.P. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Crossref PubMed Scopus (202) Google Scholar). ApoA-I with point mutations are currently being produced in order to distinguish between these possibilities.The recombinant proform of apoA-I was used in most experiments. While the properties of proapoA-I and mature apoA-I were almost identical when complexed to lipids (11Sorci-Thomas M.G. Parks J.S. Kearns M.W. Pate G.N. Zhang C. Thomas M.J. J. Lipid Res. 1996; 37: 673-683Abstract Full Text PDF PubMed Google Scholar, 12Roghani A. Zannis V.I. Biochemistry. 1988; 27: 7428-7435Crossref PubMed Scopus (15) Google Scholar, 13Westman J. Roobol C. Heymans C. Carlson L.A. Wulfert E. Scand. J. Clin. Lab. Invest. 1993; 53: 773-782Crossref PubMed Scopus (12) Google Scholar, 14McGuire K.A. Davidson W.S. Jonas A. J. Lipid Res. 1996; 37: 1519-1528Abstract Full Text PDF PubMed Google Scholar, 18Pyle L.E. Sawyer W.H. Fujiwara Y. Mitchell A. Fidge N.H. Biochemistry. 1996; 35: 12046-12052Crossref PubMed Scopus (27) Google Scholar), the ability of lipid-free mature apoA-I to promote lipid efflux was different from that of proapoA-I. We evaluated the contribution of the propeptide moiety to the facilitation of lipid efflux and reasoned that this contribution would influence equally all the carboxyl-terminally truncated forms. It cannot be totally excluded, however, that some of the effects observed with lipid-free apoA-I may be due to changes in the interaction of the propeptide with the rest of the molecule. For example, the propeptide may interact with a site at the carboxyl terminus, which could explain how it decreases the lipid-binding ability of apoA-I. Truncation to residue 150 may have eliminated this site and therefore negated the effect of the propeptide. While this possibility cannot be excluded, it is, nevertheless, unlikely; the truncation restored the activity of the mutant to the level of full-length proapoA-I, not to the level of mature apoA-I as one would expect if the mutation negated the effect of the propeptide. Alternatively, the difference in the folding of the recombinant protein, compared with the native apoA-I rather than the presence of propeptide, may have contributed to the differences observed. When complexed with phospholipid, recombinant proapoA-I and mature plasma apoA-I were equally able to facilitate cholesterol and phospholipid efflux, a phenomenon also noticed by Westman et al. (13Westman J. Roobol C. Heymans C. Carlson L.A. Wulfert E. Scand. J. Clin. Lab. Invest. 1993; 53: 773-782Crossref PubMed Scopus (12) Google Scholar).When various forms of apoA-I were incorporated into rHDL, all of them, except apoA-I-(−6-210), formed rHDL particles. Full-length apoA-I and apoA-I-(−6-222) formed mostly single-size populations, while apoA-I-(−6-150) and apoA-I-(−6-135) produced rather heterogeneous populations. All rHDL particles were of pre-β mobility. Analysis of cholesterol efflux in the presence of particles of different size should be interpreted with caution, because it has been suggested that efflux may be independently affected by the size and composition of the particles, as well as the properties of apoA-I. However, Davidson et al. (34Davidson W.S. Rodrigueza W.V. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) demonstrated that when discoidal rHDL particles are compared on the basis of their phospholipid content, other parameters such as their size, composition, and apoA-I conformation do not have a significant impact on the ability of these particles to promote cholesterol efflux. Consistent with this finding, efflux of both intracellular and plasma membrane cholesterol to rHDLs containing different mutated forms of apoA-I was similar, despite the differences in the size of the particles. However, when recalculated on a per particle basis, rHDL containing apoA-I-(−6-150) and apoA-I-(−6-135) were more active in promoting cholesterol efflux. The possible explanation for this is the presence of large rHDL particles, which on a per particle basis are better acceptors of cholesterol (34Davidson W.S. Rodrigueza W.V. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The presence of only low amounts of small particles in these preparations is unlikely to confound the findings related to cholesterol efflux, since these particles are poor cholesterol acceptors (34Davidson W.S. Rodrigueza W.V. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1995; 270: 17106-17113Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Another investigation outside the scope of this study was carried out to examine cholesterol efflux with different homogeneous subpopulations of the rHDL particles; these data will be reported elsewhere.2 A difference was expected in the intracellular cholesterol efflux between rHDL containing apoA-I-(−6-150) and apoA-I-(−6-135), a truncation eliminating a sequence that we previously suspected, from studies using monoclonal antibodies (10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar), to be associated with the ability of apoA-I to promote intracellular cholesterol efflux. No difference was observed, however. We presume that removal of a significant portion of the apoA-I affects the structural properties of the remaining peptide constraining the specific structural responsible for the intracellular cholesterol efflux. sequences associated with the efflux of intracellular cholesterol to apoA-I/phospholipid are at the of apoA-I. INTRODUCTIONIt is currently accepted that apolipoprotein A-I (apoA-I) 1The abbreviations used are: apoA-Iapolipoprotein A-IERendoplasmic reticulumrHDLreconstituted high density lipoproteinPCphosphatidylcholinePMplasma membranePOPCpalmitoyloleoyl phosphatidylcholine. -containing lipoproteins play a key role in the efflux of cellular cholesterol, the first step in the reverse cholesterol transport pathway (1Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 2Forte T.M. McCall M.R. Curr. Opin. Lipidol. 1994; 5: 354-364Crossref PubMed Scopus (80) Google Scholar, 3Barter P. Curr. Opin. Lipidol. 1993; 4: 210-217Crossref Scopus (62) Google Scholar). Thus, apoA-I-containing lipoproteins are the most efficient initial acceptors of cellular cholesterol (1Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 2Forte T.M. McCall M.R. Curr. Opin. Lipidol. 1994; 5: 354-364Crossref PubMed Scopus (80) Google Scholar), and removal of these lipoproteins from the plasma greatly reduces the release of cholesterol from cells (4Fielding C.J. Moser K. J. Biol. Chem. 1982; 257: 10955-10960Abstract Full Text PDF PubMed Google Scholar). The mechanism of how apoA-I executes this function remains, however, unresolved. It has been demonstrated that short peptides mimicking the secondary structure of apoA-I are able, to a certain extent, to imitate its ability to facilitate removal of cellular cholesterol (5Davidson W.S. Lund-Katz S. Johnson W.J. Anantharamaiah G.M. Palgunachari M.N. Segrest J.P. Rothblat G.H. Phillips M.C. J. Biol. Chem. 1994; 269: 22975-22982Abstract Full Text PDF PubMed Google Scholar, 6Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. J. Clin. Invest. 1994; 94: 1698-1705Crossref PubMed Scopus (175) Google Scholar, 7Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar), suggesting that a rather nonspecific mechanism, which relies mainly on the ability of apoA-I to bind lipids, appears to be involved. On the other hand, new evidence suggests that specific sequences of apoA-I are responsible for cholesterol efflux (8Banka C.L. Black A.S. Curtiss L.K. J. Biol. Chem. 1994; 269: 10288-10297Abstract Full Text PDF PubMed Google Scholar, 9Fielding P.E. Kawano M. Catapano A.L. Zoppo A. Marcovina S. Fielding C.J. Biochemistry. 1994; 33: 6981-6985Crossref PubMed Scopus (85) Google Scholar, 10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar), pointing to the involvement of a more specific and possibly receptor-dependent mechanism.In our previous work using monoclonal antibodies against specific epitopes of apoA-I, we identified a sequence, apoA-I-(140-150), that was associated with the ability of apoA-I to promote efflux of intracellular cholesterol (10Sviridov D. Pyle L. Fidge N. Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar). In order to further investigate the involvement of various domains of apoA-I in cholesterol efflux, we used recombinant proapoA-I, either intact or truncated, in the lipid-free form or incorporated into reconstituted high density lipoprotein (rHDL), to study the effect of the carboxyl-terminal domain truncations on the efflux of cholesterol and phospholipid from cultured cells. While the proform of apoA-I is a minor component of circulating apoA-I, its known properties do not deviate strongly from mature apoA-I (11Sorci-Thomas M.G. Parks J.S. Kearns M.W. Pate G.N. Zhang C. Thomas M.J. J. Lipid Res. 1996; 37: 673-683Abstract Full Text PDF PubMed Google Scholar, 12Roghani A. Zannis V.I. Biochemistry. 1988; 27: 7428-7435Crossref PubMed Scopus (15) Google Scholar, 13Westman J. Roobol C. Heymans C. Carlson L.A. Wulfert E. Scand. J. Clin. Lab. Invest. 1993; 53: 773-782Crossref PubMed Scopus (12) Google Scholar, 14McGuire K.A. Davidson W.S. Jonas A. J. Lipid Res. 1996; 37: 1519-1528Abstract Full Text PDF PubMed Google Scholar) and the biological principles underlying structure-function relationships between cholesterol efflux and proteins should be equally relevant for mature apoA-I and proapoA-I.