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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1637-1643.)
© 1997 American Heart Association, Inc.

Articles

Evidence That Apolipoprotein A-I_Milano Has Reduced Capacity, Compared With Wild-Type Apolipoprotein A-I, to Recruit Membrane Cholesterol

John K. Bielicki; Mark R. McCall; Lori J. Stoltzfus; Amir Ravandi; Arnis Kuksis; Edward M. Rubin; ; Trudy M. Forte

From the Life Sciences Division 1-213, Department of Molecular and Nuclear Medicine, Ernest Orlando Lawrence Berkeley National Laboratory, University of California at Berkeley.

Correspondence to John K. Bielicki, Life Sciences Division 1-213, Department of Molecular and Nuclear Medicine, Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720.

	Abstract

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Abstract Human carriers of apolipoprotein (apo) A-I_Milano are heterozygous for an Arg₁₇₃Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-I_Milano may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-I_Milano to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-I_Milano and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (2 µg/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (d=1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66±12%) of the lipidated apoA-I_Milano was in the homodimer form. Compositional analyses revealed that apoA-I_Milano nascent HDL had a significantly lower (P<.001) unesterified cholesterol/phospholipid mole ratio (0.47±0.10) than wild-type apoA-I complexes (1.29±0.14), indicating that apoA-I_Milano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-I_Milano nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-I_Milano activated LCAT normally. We conclude that, even though apoA-I_Milano is a normal activator of LCAT, it is less efficient than wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-I_Milano may be unrelated to the initial stages of reverse cholesterol transport.

Key Words: nascent HDL assembly • apoA-I_Milano • cellular cholesterol recruitment • reverse cholesterol transport • LCAT activation

	Introduction

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Elevated plasma concentrations of HDL are associated with reduced risk of atherosclerosis.^{1 2 3} The protective effects of HDL are due in part to its ability to promote the efflux of cholesterol from extrahepatic cells and mediate the transport of cholesterol to the liver for catabolism.^{4 5 6 7} ApoA-I, the major structural protein of HDL, is thought to facilitate this reverse cholesterol transport process by serving as cofactor for LCAT. The esterification of cholesterol on HDL catalyzed by LCAT favors the net efflux of cellular cholesterol and enables cholesteryl esters to accumulate in HDL for intravascular transport.

Recent experimental evidence indicates that apoA-I, in lipid-free form, can recruit phospholipid and cholesterol from cells (including CHO cells, fibroblasts, and macrophage-derived foam cells), thus forming lipid complexes in the medium.^{8 9 10 11 12 13} We have previously shown that the recruitment of membrane lipids from CHO cells by apoA-I results in the assembly of discrete nascent HDL subpopulations that have the following characteristics: (1) discoidal morphology,⁸ (2) elevated UC/PL molar ratios (compared with plasma HDL),⁹ and (3) pre- electrophoretic mobility in agarose.¹⁰ These features are similar to the physical/chemical properties of specific HDL subpopulations found in human plasma and interstitial fluid,^{14 15 16 17} suggesting that in vivo, the extracellular assembly of nascent HDL may reflect the initial stages of reverse cholesterol transport, in which apoA-I mediates the removal of cholesterol from peripheral cells. Nascent HDL generated on the association of apoA-I with membrane lipids is also an efficient substrate for LCAT, yielding transformation products similar in size and composition to plasma HDL.¹⁰

ApoA-I is a 243–amino acid protein; the most striking feature of the apoA-I secondary structure is the presence of tandem, 22–amino acid amphipathic -helical lipid-binding domains located in the carboxy-terminal region of the molecule.^{18 19} The cooperative interactions between these lipid-binding domains are thought to determine HDL particle size and composition.^{20 21} Moreover, specific -helical domains have also been implicated in LCAT activation²² and cellular cholesterol efflux.²³

The apoA-I_Milano mutation consists of an Arg₁₇₃Cys substitution within one of the carboxy-terminal -helical domains of apoA-I.^{24 25} In human subjects, all of whom are heterozygous, this mutation produces an HDL deficiency in which small, dense HDL₃ subpopulations predominate.^{26 27} The presence of the cysteine residue permits dimerization of apoA-I_Milano; thus, in human apoA-I_Milano carriers, a large proportion of HDL contains apoA-I_Milano homodimers and apoA-I_Milano/A-II heterodimers.^{24 26 27 28} The dimerization of apoA-I_Milano is thought to restrict HDL particle size and, by doing so, impair LCAT–mediated conversion of HDL₃ to HDL₂. Because human subjects are heterozygous for the apoA-I_Milano mutation, the presence of wild-type apoA-I has made it difficult to directly evaluate the role of apoA-I_Milano in HDL cholesterol metabolism. In the present study, we used a well-characterized cell-culture model for HDL assembly to assess the ability of apoA-I_Milano to recruit cell cholesterol. The capacity of apoA-I_Milano nascent HDL to serve as a substrate for LCAT was also evaluated to establish whether, in the absence of wild-type apoA-I, apoA-I_Milano is an efficient activator of LCAT.

	Methods

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Transfection of CHO K1 Cells With the Gene for ApoA-I_Milano and Isolation of High-Expressing Clones
The Arg₁₇₃Cys substitution was prepared (Mutagene kit, Bio-Rad) in a 2.2-kb Sma I fragment of the human apoA-I gene. DNA sequencing was performed to verify the mutation (data not shown). The apoA-I_Milano gene was inserted into the pcDNA3 expression vector. This vector contains the cytomegalovirus promoter/enhancer region, which is constitutively expressed at high levels in most mammalian cells. CHO K1 cells were stably transfected with the apoA-I_Milano DNA construct by electroporation, and clonal cell lines were prepared by infinite-dilution methodology. High-expressing clones were identified by Western blot analysis of conditioned medium with polyclonal antisera specific for wild-type apoA-I. CHO K1 cells expressing the gene for wild-type, human apoA-I have been previously described.⁸

Isolation of Nascent HDL From CHO-Conditioned Medium
CHO cells expressing the gene for apoA-I_Milano were maintained in 175-cm² culture flasks (Corning Costar Corp) in McCoy's 5A medium containing 10% Fetal Clone II (Hyclone Laboratories) in an atmosphere of 5% CO₂/95% air at 37°C. For experiments, clonal cell lines expressing the gene for either wild-type apoA-I or apoA-I_Milano were grown in 375-cm² flasks (30 flasks per experiment). Confluent monolayers were rinsed three times with sterile Hank's balanced salt solution (20 mL per flask). A fourth rinse with serum-free McCoy's 5A medium (43 mL per flask) was extended overnight to ensure that flasks were completely rinsed of all lipoproteins from original growth medium. For evaluations of nascent HDL assembly, cell monolayers were incubated with serum-free McCoy's 5A medium (43 mL per flask) for 24 hours; this incubation time has been shown to be sufficient for maximal recruitment of membrane lipids by apoA-I.^{8 9 10 11} Conditioned medium was harvested and cooled to 4°C. Floating cells were pelleted by low-speed centrifugation (1000g), and EDTA (2.7 mmol/L), PMSF (0.5 mmol/L), and gentamicin (50 µg/mL) were added to medium supernatant. Media were concentrated 100-fold using a Minitan ultrafiltration system (Millipore Corp) fitted with a cellulose membrane (10 000 molecular weight cutoff). Nascent HDL (d=1.063 to 1.21 g/mL) was isolated by sequential preparative ultracentrifugation.²⁹ Samples were dialyzed against 0.15 mmol/L NaCl/2.7 mmol/L EDTA (saline-EDTA) for physical/chemical characterizations and against 20 mmol/L Tris (pH 8.0)/saline-EDTA for LCAT reactivity studies.

Quantification of Apolipoprotein Secretion From CHO Clonal Cell Lines
Apolipoprotein mass in concentrated medium was measured by radial immunodiffusion by using polyclonal antisera against wild-type apoA-I.³⁰ The lower limit of detection was 25 ng. Cellular protein was measured by the method of Markwell et al.³¹ Secretion rates were expressed as micrograms apolipoprotein per milligram cell protein per 24 hours.

Physical and Chemical Properties of ApoA-I_Milano Nascent HDL
Nascent HDL protein composition was evaluated by using 4% to 20% SDS-PAGE gels according to the method of Laemmli.³² Proteins were visualized either by Coomassie R-250 stain or by Western blot analysis using monoclonal antibodies (Chemicon) against wild-type apoA-I; the distribution of monomeric and dimeric forms of apoA-I_Milano were determined by densitometric scanning of Coomassie-stained gels. Nondenaturing gradient (4% to 30%) gel electrophoresis was used to evaluate nascent HDL particle size distribution.³³ Agarose gel electrophoresis was performed to determine relative charge properties of nascent HDL; Paragon Lipogels were used (Beckman). Protein content of nascent HDL was measured using a protein reagent following the manufacturer's (Bio-Rad Laboratories) instructions. Cholesterol was measured by the method of Sale et al³⁴ and phospholipid was quantified as described by Chen et al.³⁵

LCAT Reactivity of ApoA-I_Milano Nascent HDL
LCAT was isolated from normolipidemic human plasma by the method of Chen and Albers.³⁶ Purity was assessed by SDS-PAGE, and a single band corresponding to the molecular weight of LCAT (66 Kd) was observed. Nascent HDL (0.15 mg UC per milliliter) was incubated (4°C) with [¹⁴C]cholesterol-labeled LDL (0.3 mg UC per milliliter) for 48 hours to allow equilibration of [¹⁴C]cholesterol with nascent HDL. Specific activity of [¹⁴C]cholesterol was 1 µCi/mg LDL UC. Rates of [¹⁴C]cholesterol esterification were evaluated in reaction mixtures (0.25 mL final volume) containing 0.15 mg protein per milliliter of nascent HDL, 0.75 mg protein per milliliter of LDL, 1.5% human serum albumin, 50 µg gentamicin per milliliter, and sufficient purified LCAT to esterifiy 7.0 nmol cholesterol per hour. Reactions were incubated at 37°C for up to 24 hours. At specified times, samples were removed, cooled to 4°C, and lipids were extracted with hexane. Cholesterol and cholesteryl esters were separated by instant TLC, using fiberglass sheets coated with silica (Gelman Sciences Inc). Results were expressed as a percentage of the initial [¹⁴C]cholesterol converted to [¹⁴C]cholesteryl ester.

To determine whether nascent HDL particles were the major site of the LCAT reaction, we performed a control experiment in which LDL was omitted from LCAT incubations and the mass of cholesteryl ester associated with nascent HDL was quantified. We found that in the absence of LDL, a similar percentage of cholesterol esterification was obtained with apoA-I_Milano nascent HDL compared with particles incubated with LDL. These observations indicated that apoA-I_Milano was indeed able to activate LCAT (data not shown) and the esterification of cholesterol was occurring on nascent HDL particles.

In one experiment, the particle size distribution of LCAT transformation products was evaluated. LCAT reactions were carried out (24 hours) as described above, except [¹⁴C]cholesterol was omitted from reaction mixtures; control incubations (24 hours) were run in the absence of LCAT enzyme. Immediately after incubations, HDL (d=1.063 to 1.21 g/mL) was isolated by sequential ultracentrifugation and the particle size distribution determined by nondenaturing gradient (4% to 30%) gel electrophoresis followed by Western blot analysis.

	Results

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Secretion and Lipidation of ApoA-I_Milano
CHO clonal cell lines expressing the genes for either apoA-I_Milano or wild-type apoA-I were incubated with serum-free medium for 24 hours. Quantification of apolipoprotein mass in conditioned medium revealed that both apolipoproteins accumulated to the same extent (1.9±0.8 and 1.7±0.7 µg/mg cell protein per 24 hours for apoA-I_Milano and wild-type apoA-I, respectively; n=7).

Previously we have shown that wild-type apoA-I can recruit membrane lipids to form nascent HDL.^{8 9 10} To determine the relative proportion of apoA-I_Milano compared with wild-type apoA-I to form lipid complexes, the percentage of medium apolipoprotein associated with nascent HDL was quantified. We found that a similar amount of each apolipoprotein floated at d=1.063 to 1.21 g/mL (apoA-I_Milano=15±5% and wild-type apoA-I=14±2%; n=7), indicating that apoA-I_Milano was able to recruit membrane lipids for the assembly of nascent HDL particles.

Chemical Composition of ApoA-I_Milano Nascent HDL
The chemical compositions of apoA-I_Milano and apoA-I nascent HDL are shown in the Table. Both complexes exhibited similar weight distributions of protein and lipid (35% protein versus 65% lipid), consistent with their isolation at d=1.063 to 1.21 g/mL. Marked differences, however, were observed in lipid composition. ApoA-I_Milano nascent HDL possessed relatively more (20%) phospholipid but less (56%) cholesterol than wild-type apoA-I nascent HDL. These changes in lipids produced a 64% reduction in the UC/PL mole ratio (0.47±0.10 versus 1.29±0.14 for apoA-I_Milano and wild-type apoA-I, respectively), indicating that apoA-I_Milano has a limited capacity to recruit membrane cholesterol.

View this table: [in this window] [in a new window]   Table 1. Chemical Composition of Nascent HDL Isolated From Conditioned Medium of CHO Cells Expressing Either Wild-Type ApoA-I or ApoA-IMilano

ApoA-I_Milano Dimerization on Nascent HDL
Because the Arg₁₇₃Cys substitution can produce apoA-I_Milano dimerization, we evaluated the extent of apoA-I_Milano dimerization on nascent HDL complexes. SDS-PAGE (Fig 1) revealed that a majority (66±12%, n=7) of the apoA-I_Milano on nascent HDL was in the dimeric form. Fig 1, lane 5, shows that on reduction with ß-mercaptoethanol, only a single band, corresponding in molecular weight (28 Kd) to wild-type apoA-I, was observed, consistent with the behavior of the cysteine substitution within the apoA-I primary sequence.

View larger version (56K): [in this window] [in a new window]   Figure 1. Dimerization of apoA-IMilano on nascent HDL determined by SDS-PAGE. Nascent HDL (d=1.063 to 1.21 g/mL) was obtained from conditioned medium of CHO cells expressing either apoA-IMilano or wild-type apoA-I. Proteins were separated by SDS-PAGE and transferred to nitrocellulose sheets for Western blot analysis, using monoclonal antibodies against wild-type apoA-I. Lane 1 contains molecular weight standards; lane 2, lipid-free apoA-I; lane 3, apoA-I nascent HDL; lane 4, apoA-IMilano nascent HDL; and lane 5, apoA-IMilano nascent HDL plus 5 mmol/L ß-mercaptoethanol.

Analysis of Nascent HDL Particle Size
The effect of apoA-I_Milano on nascent HDL particle size was evaluated by nondenaturing gradient gel electrophoresis. Densitometric scans of Coomassie-stained gels (Fig 2) showed that the apoA-I_Milano particle size distribution was skewed to smaller particles (7.4 nm) compared with wild-type apoA-I, in which larger-sized (11- and 9-nm) particles predominated. Previous studies with wild-type apoA-I indicated that the formation of 11- and 9-nm particles was dependent on the apolipoprotein concentration,⁹ whereas 2 to 4 µg/mL generated mostly 7.4-nm particles. To establish that the predominance of 7.4-nm particles in apoA-I_Milano medium was the consequence of the apoA-I_Milano mutation and thus independent of concentration, nontransfected CHO cells were incubated with up to 10 µg/mL lipid-free apoA-I_Milano. At all concentrations tested (from 2 to 10 µg/mL), the 7.4-nm particle was a major assembly product (data not shown), indicating that the Arg₁₇₃Cys substitution was most likely responsible for the restriction in nascent HDL particle size.

View larger version (7K): [in this window] [in a new window]   Figure 2. Nascent HDL particle size distribution. ApoA-IMilano and apoA-I nascent HDL (d=1.063 to 1.21 g/mL) particle size distributions were determined by nondenaturing gradient gel (4% to 30%) electrophoresis. Scans of a representative Coomassie G-250–stained gel are shown. Numbers over peaks indicate particle diameters (nanometers). Protein loads were 5 µg per well.

Agarose Gel Electrophoresis of ApoA-I_Milano Nascent HDL
Like wild-type apoA-I complexes, apoA-I_Milano nascent HDL was found to migrate to the pre- position on agarose gels (Fig 3). Moreover, its cathodal migration was somewhat greater than wild-type apoA-I nascent HDL; this more rapid mobility is consistent with the loss of a positively charged arginine residue due to the Arg₁₇₃Cys substitution.

View larger version (34K): [in this window] [in a new window]   Figure 3. Agarose gel electrophoresis of apoA-IMilano nascent HDL. Relative charge properties of apoA-IMilano nascent HDL were evaluated by agarose gel electrophoresis; proteins were transferred to nitrocellulose sheets and probed with monoclonal antibodies to apoA-I. Lane 1 contains lipid-free apoA-I (pre-ß mobility, 0.3 µg protein per well); lane 2, plasma HDL ( mobility, 0.6 µg protein per well); lane 3, apoA-I nascent HDL (pre-, 0.5 µg protein per well); and lane 4, apoA-IMilano nascent HDL (pre-, 0.9 µg protein per well).

LCAT Reactivity of ApoA-I_Milano Nascent HDL
In human apoA-I_Milano carriers, dimerization of apoA-I_Milano is characterized by a reduction in cholesterol esterification and the presence of primarily small, dense HDL.²⁷ Critical analysis of these alterations is confounded by the presence of wild-type apoA-I and apoA-II in the plasma of such patients. It is well known that nascent HDL is an efficient substrate for LCAT and represents a precursor to mature plasma HDL. Therefore, we reasoned that by examining the LCAT reactivity of apoA-I_Milano nascent HDL, we could directly assess whether this molecular variant could activate LCAT normally. As shown in Fig 4, apoA-I_Milano complexes had a [¹⁴C]cholesterol esterification rate similar to wild-type apoA-I complexes, indicating that the mutant protein had a normal capacity to activate LCAT.

View larger version (20K): [in this window] [in a new window]   Figure 4. Esterification of [14C]cholesterol on apoA-IMilano nascent HDL by LCAT. ApoA-IMilano () and apoA-I nascent HDL () were incubated with purified LCAT and the esterification of [14C]cholesterol was evaluated as described in "Methods." At the times indicated, samples were removed and lipids extracted with hexane. Cholesterol and cholesteryl esters were separated by TLC, and radioactivity associated with each was quantified by liquid scintillation spectroscopy. Results are expressed as a percentage of the initial [14C]cholesterol converted to [14C]cholesteryl ester. Values are mean±SD; n=4.

The effect of cholesterol esterification on nascent HDL transformation into mature particles was determined by nondenaturing gradient gel electrophoresis. Fig 5 reveals that there were major differences in the response of apoA-I_Milano complexes compared with those of wild-type apoA-I. As expected, the apoA-I_Milano nascent HDL incubated without LCAT contains small 7.3-nm particles (not seen in the normal profile) together with particles in the region corresponding to 9.0 nm. After incubation with LCAT, the 7.3-nm apoA-I_Milano complex disappears and several new larger-sized (8.6-, 11.0-, 12.5-, and 15.0-nm) particles were generated. In contrast, wild-type apoA-I complexes yielded primarily 8.6-nm particles on LCAT treatment; the size of this transformation product is similar to the major apoA-I–containing HDL isolated from human plasma. These data indicate that although apoA-I_Milano could activate LCAT normally, the product distribution resulting from cholesterol esterification was abnormal. The latter may be explained in part by fusion of the smaller 7.4-nm particles with the 8.6-nm LCAT transformation product, thus yielding larger-sized particles; the incremental change (2.5 nm) in particle diameter going from the 8.6 to 11.0 and 12.5 to 15.0 nm may reflect the addition of an apoA-I_Milano homodimer to the surface of these particles.

View larger version (66K): [in this window] [in a new window]   Figure 5. Particle size distribution of LCAT transformation products. ApoA-IMilano and apoA-I nascent HDL were incubated with purified LCAT as described in "Methods." LCAT transformation products (d=1.063 to 1.21) were isolated by sequential preparative ultracentrifugation. Nondenaturing gradient gel electrophoresis was performed to separate HDL subpopulations, and particles were transferred to nitrocellulose sheets and probed with antibody to apoA-I. Lane 1 contains molecular weight standards of known diameters (in nm): thyroglobulin 17.0, ferritin 12.2, catalase 9.5, lactate dehydrogenase 8.2, and albumin 7.1. Lane 2 shows wild-type apoA-I nascent HDL incubated (24 hours, 37°C) without LCAT; lane 3, LCAT transformation products obtained with apoA-I nascent HDL; Lane 4, apoA-IMilano nascent HDL incubated without LCAT; and lane 5, LCAT transformation products obtained with apoA-IMilano nascent HDL.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates, for the first time, that apoA-I_Milano has a limited capacity to recruit membrane cholesterol compared with wild-type apoA-I. In addition, nascent HDL formed by the association of apoA-I_Milano with membrane lipids was predominantly small 7.4-nm particles rather than the larger 9- and 11-nm complexes formed by wild-type apoA-I. Taken together, these observations indicate that the Arg₁₇₃Cys substitution in the apoA-I primary sequence interfered with the normal process of cellular cholesterol recruitment and nascent HDL assembly.

We have previously shown that the 11- and 9-nm particles formed on incubation of wild-type apoA-I with CHO cells possessed a relatively high UC/PL mole ratio (0.92 and 0.89, respectively) compared with the UC/PL mole ratio (0.40) of the small 7.4-nm particle.^{8 9 10} Thus, the formation of larger complexes is indicative of efficient removal of cellular cholesterol. ApoA-I_Milano, unlike wild-type apoA-I, formed only small quantities of larger complexes, suggesting that the mutation is associated with a decreased efficiency for cholesterol removal from cells; this was corroborated by the low UC/PL mole ratio (0.47) of apoA-I_Milano nascent HDL compared with wild-type apoA-I complexes (1.29).

The most striking structural change attributed to the Arg₁₇₃Cys substitution is the dimerization of apoA-I_Milano. Since a majority of apoA-I_Milano associated with nascent HDL was in the dimeric form and as the particle size distribution was skewed to small 7.4-nm complexes, it is likely that dimerization of apoA-I_Milano contributed to the restriction of nascent HDL particle size. The introduction of an interchain disulfide bridge may "lock" the conformation of the protein such that small, extremely stable particles are generated. In its locked form, accumulation of additional cholesterol may be minimized.

The Arg₁₇₃Cys substitution also reduces the lipid-binding affinity of the monomeric form of apoA-I_Milano compared with wild-type apoA-I³⁷ ; reduced lipid-binding affinity could very well limit the ability of apoA-I_Milano to recruit membrane lipids. We have previously demonstrated that the small (7.4-nm), wild-type apoA-I particles are extremely stable on reincubation with CHO cells; addition of lipid-free apoA-I was required to drive the formation of larger-sized (11- and 9-nm) lipid complexes.³⁸ Although speculative, the monomeric form of apoA-I_Milano, with its reduced lipid-binding affinity, may be unable to destabilize the small 7.4-nm particles to drive the formation of larger-sized lipid complexes; thus, relatively smaller (7.4-nm), cholesterol-poor particles accumulate in the medium.

Another mechanism that could account for the low cholesterol content of apoA-I_Milano nascent HDL may be related to impaired ability of apoA-I_Milano to recruit specific phospholipid subclasses. Preliminary experiments have in fact revealed major differences in the phospholipid composition of apoA-I_Milano nascent HDL compared with wild-type apoA-I particles. Wild-type apoA-I nascent HDL was composed mostly of phosphatidylcholine (78% of total phospholipid mass) and to a lesser extent sphingomyelin (15%), phosphatidylethanolamine (6.5%), and phosphatidylinositol (3.5%). In contrast, apoA-I_Milano nascent HDL exhibited a relative abundance of acidic phospholipids, as well as dramatically increased phosphatidylethanolamine, as shown by the following mass distributions: phosphatidylcholine, 13%; sphingomyelin, 11%; phosphatidylethanolamine, 40%; phosphatidylinositol, 20%; and phosphatidylserine, 8%. These alterations in phospholipid composition suggest differences in the site (ie, specific membrane domains) of lipid recruitment. Phosphatidylserine and phosphatidylinositol are most abundant on the inner leaflet of the plasma membrane, indicating that apoA-I_Milano may penetrate more deeply into the plasma membrane, destabilizing its bilayer structure, and thereby enabling access to the cytoplasmic face of the membrane; wild-type apoA-I nascent HDL, on the other hand, exhibited a predominance of phosphatidylcholine and sphingomyelin, indicative of recruitment from the outer leaflet of the plasma membrane. These observations suggest that the low UC/PL mole ratio of apoA-I_Milano nascent HDL compared with wild-type apoA-I may in part reflect the cholesterol content of the specific membrane domains with which these apolipoproteins interact.

In normal subjects, LCAT-mediated esterification of cholesterol is associated with the transformation of small, dense HDL to larger, more buoyant HDL. Plasma of apoA-I_Milano carriers, however, is characterized by a deficiency of HDL and the predominance of smaller, more dense HDL particles.^{24 25 26 27} It has been suggested that dimerization of apoA-I_Milano impairs cholesterol esterification, thus accounting for the abnormal distribution of HDL particles in such subjects.²⁷ Studies on plasma of apoA-I_Milano subjects are confounded by the presence of wild-type apoA-I and apoA-I_Milano/apoA-II heterodimers, making it difficult to directly assess the ability of apoA-I_Milano to activate LCAT. In the present study, we found that the rate of cholesterol esterification on apoA-I_Milano nascent HDL was identical to that of wild-type complexes, strongly suggesting that the mutation did not alter LCAT activation. These in vitro studies further suggest that in human apoA-I_Milano carriers, LCAT has the potential to be fully activated.

Regardless of its normal capacity to activate LCAT, apoA-I_Milano, by reason of its dimerization, could very well limit cholesteryl ester formation by imposing a steric constraint, which restricts HDL particle size. Paradoxically, in our in vitro system, apoA-I_Milano yielded mostly larger-sized 11.0-, 12.5-, and 15.0-nm LCAT transformation products compared with wild-type apoA-I, in which 8.6-nm particles were abundant. As discussed earlier, these larger products may result from particle fusion during the transformation process. In human apoA-I_Milano carriers, however, HDL particle size is very much restricted, indicating that factors other than apoA-I_Milano homodimers restrict HDL to small, dense HDL. In vivo, a substantial amount of apoA-I_Milano is in the form of apoA-I_Milano/apoA-II heterodimers.²⁶ Although speculative, the presence of these heterodimers on HDL may impose the major structural constraint that restricts HDL particle size, thereby limiting cholesterol esterification in vivo.

Despite alterations in HDL cholesterol metabolism, apoA-I_Milano subjects appear to be resistant to the development of coronary heart disease, which has led to the suggestion that apoA-I_Milano may have antiatherogenic properties.³⁹ Roma et al⁴⁰ have demonstrated increased catabolism of apoA-I_Milano, suggesting a rapid clearance of HDL from the circulation. The hypercatabolism of apoA-I_Milano HDL is thought to facilitate the transport of cholesterol from peripheral tissues to sites of cholesterol catabolism and/or utilization (liver and/or steroidogenic cells). At this time, however, information regarding the fate of HDL cholesteryl ester in apoA-I_Milano subjects is unavailable. Therefore, whether the accelerated catabolism of apoA-I_Milano is truly accompanied by increased transport of cholesterol remains controversial. The reduced capacity of apoA-I_Milano HDL to support LCAT-mediated cholesterol esterification in vivo suggests that a limited amount of HDL cholesteryl ester would be available for delivery to hepatic and steroidogenic cells by the "selective uptake" mechanism. Similarly, HDL cholesteryl ester would be unavailable for the cholesteryl ester transfer protein–mediated transfer of cholesteryl esters to apoB-containing lipoproteins. Moreover, our finding that apoA-I_Milano has limited capacity to recruit cellular cholesterol suggests that the antiatherogenic properties attributed to this molecular variant may be unrelated to reverse cholesterol transport.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein CHO = Chinese hamster ovary LCAT = lecithin:cholesterol acyltransferase PAGE = polyacrylamide gel electrophoresis UC/PL = unesterified cholesterol/phospholipid

	Acknowledgments

 
This work was supported by NIH program project grant HL 18574 from the National Heart, Lung, and Blood Institute; National Research Service Award training grant HL 07279 (to L.J. Stoltzfus; and a research fellowship (to J.K. Bielicki) from the American Heart Association, California affiliate. The work was conducted at the Ernest Orlando Lawrence Berkeley National Laboratory through the US Department of Energy under contract No. DE-AC03-76SF00098. The excellent technical assistance of Un Hui Har and Laura Knoff is greatly appreciated.

Received September 12, 1996; accepted January 24, 1997.

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