©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Apolipoprotein A-I in Cholesterol Transfer between Lipoproteins
EVIDENCE FOR INVOLVEMENT OF SPECIFIC apoA-I DOMAINS (*)

Qiang-Hua Meng , Jean Bergeron (§) , Daniel L. Sparks , Yves L. Marcel (¶)

From the (1) Lipoproteins and Atherosclerosis Group, University of Ottawa Heart Institute and the Departments of Pathology and Laboratory Medicine and of Biochemistry, University of Ottawa, Ottawa, Ontario, K1Y 4E9, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A series of monoclonal antibodies against epitopes spanning different domains of apoA-I have been tested for their effects on unesterified cholesterol transfer between low density lipoprotein (LDL) and well-defined homogenous lipoproteins reconstituted with phosphatidylcholine, cholesterol, and apoA-I (LpA-I). Antibodies 2G11 (reacting between residues 25 and 110), A05 (residues 25-82), A03 (residues 135-140), A44 and r5G9 (residues 149-186), and 4A12 (residues 173-205) significantly inhibit cholesterol transfer from LDL to Lp2A-I while they enhance transfer in the opposite direction, thus causing an increased net transfer to LDL. Most of these monoclonal antibodies (mAbs) also enhance phospholipid transfer to LDL but in a lesser and variable proportion relative to cholesterol. Their epitopes are mainly contained within domains that are predicted to be amphipathic -helices. In contrast, mAbs 4H1 (residues 2-8), 3G10 (residues 96-121), and 5F6 (residues 116-141) have little or no effect on either cholesterol or phospholipid transfer, and the epitopes for these three mAbs have been shown in earlier studies to be structurally and functionally related. Their immunoreactivity responds similarly to variation in lipoprotein cholesterol content, and the antibodies binding to these sites compete with one another and have similar effects on the cholesterol esterification reaction. Thus, the current results are compatible with the hypothesis that they form an integrated domain with a common function in cholesterol metabolism, possibly as part of a hinge domain. Most mAbs were found to increase significantly the -helicity of apoA-I in the Lp2A-I immunecomplexes, suggesting that they may increase the stability of the lipid-bound apoA-I. However, not unexpectedly, there is no correlation between the effects of mAbs on -helicity and their effects on cholesterol or phospholipid transfer since each mAb has a discrete effect on these transfers. These studies demonstrate the specificity of LpA-I particles in cholesterol transport and document the existence of apoA-I domains with different functions in cholesterol transport.


INTRODUCTION

The efflux of unesterified (free) cholesterol from cells to acceptor lipoproteins has been well studied and appears to be satisfactorily explained by the mechanism of aqueous diffusion (for review, see Ref. 1). However apolipoprotein A-I-containing lipoproteins (LpA-I)() with a pre migration are the most avid acceptors of cellular cholesterol, and the specificity of these lipoproteins in this pathway also suggests that greater binding (or affinity) of cholesterol with these lipoproteins provides a directionality for cholesterol flux (2, 3) . In contrast, the transfer of cholesterol between lipoproteins appears to proceed with a different specificity as demonstrated by the transfer from LDL to plasma lipoproteins, which is first channeled through -migrating lipoproteins rather than pre-lipoproteins (4, 5) . Therefore, the observed preferential binding affinity of pre LpA-I for cholesterol depends on the type of donor and acceptor involved (the transfer interface). Such a selectivity of acceptors and interfacial systems suggest that a complex mechanism is involved, which may include both an activation step, possibly a desorption step as suggested by McLean and Phillips (6) or a unimolecular activation event, perhaps the movement of cholesterol to a more exposed (hydrated) position, and a collision step (7) characterized by a directionality specific for each interface, such as cells to lipoproteins and lipoproteins to lipoproteins.

We propose that the specificity of the collision at the interface must depend on an apolipoprotein specificity that implicates the interaction of defined and possibly different domains with each donor and acceptor system. The complementarity of such domains, which may depend on charge (8, 9) , would differ as a function of the interface involved. This hypothesis can be tested using monoclonal antibodies against apoA-I as probes to modify the interactions between the LpA-I and other cholesterol-carrying structures. We have selected LDL and discoidal LpA-I particles as a simpler and well defined system (10, 11, 12, 13, 14) , compared with the cell-lipoprotein interface, and have used a selection of mAbs reacting with epitopes distributed over most of apoA-I sequence (15) to test the directionality of the interaction between these two types of lipoproteins. The results obtained in this study are compatible with a concept of directional specificity in the interaction of lipoproteins but more importantly demonstrate the involvement of specific domains of apoA-I in the control of cholesterol binding and desorption. However the unidirectional effect of the active mAbs on cholesterol transfer and their increase of the -helicity of several Lp2A-I immunecomplexes suggest that the action of mAbs in cholesterol transfer is more complex than a simple steric hindrance and is probably related to specific conformational modifications.


MATERIALS AND METHODS

Isolation of Plasma LDL, HDL, and Purification of apoA-I

Plasma LDL was prepared from pooled plasma from normolipemic volunteers by sequential flotation ultracentrifugation as described previously (16) . The LDL fraction (KBr density = 1.020-1.063 g/ml) of plasma was dialyzed against Tris-EDTA buffer containing 10 m M Tris-HCl, pH 8.0, 1 m M EDTA, and 1 m M NaNand stored at 4 °C under Nfor up to 2 weeks. The HDL fraction (KBr density = 1.063-1.21 g/ml) of plasma was delipidated, resolubilized in 6 M guanidine-HCl, and chromatographed on two serial Sephacryl S-200 gel filtration columns (100 2.5 cm) equilibrated with 3 M guanidine in 10 m M Tris buffer, pH 8.0, to isolate apoA-I protein (17) .

Reconstitution and Purification of Homogeneous LpA-I Particles

Reconstituted HDL was prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) (Sigma), cholesterol (Sigma), and apoA-I at initial molar ratio of 120:6:1 or 88:44:1 by cholate dialysis following previously published procedures (18, 19) . Homogeneous lipoprotein particles containing two, three, or four apoA-I molecules (Lp2A-I, -3A-I, or -4A-I) were purified by gel filtration on two serial agarose Bio-Gel 5 M columns (95 2.5 cm) (Bio-Rad) equilibrated with Tris-EDTA buffer. The Lp2A-I (9.6 nm), Lp3A-I (13.4 nm), and Lp4A-I (17.0 nm) particles were purified from the above reconstituted HDL at an initial molar ratio of 120:6:1, and Lp2A-I (7.8 nm), Lp2A-I (8.6 nm), and Lp3A-I (10.8 nm) particles were purified from the above reconstituted HDL at an initial molar ratio of 88:44:1. The Lp2A-I (10.2 nm) particles were prepared according to Sparks et al. (20) at an initial POPC/cholesterol/apoA-I molar ratio of 150:6:1. The characteristics of these particles were well defined and reported in previous studies (21) . Size characterization of LpA-I particles was done by native gradient gel electrophoresis using high molecular calibration kit (Pharmacia Biotech Inc.) with reference globular proteins (thyroglobulin, 17.0 nm; ferritin, 12.2 nm; lactate dehydrogenase, 8.16 nm; and albumin, 7.1 nm) as described by others (22) . The number of apoA-I molecules/LpA-I particles was determined by the cross-linking with dimethyl suberimidate technique (23) . The protein concentration of lipoproteins was measured by Lowry assay (24) , and that of cholesterol and POPC were measured by enzymatic kits (Boehringer Mannheim).

Labeling of LDL and LpA-I

To measure cholesterol transfer from LDL to LpA-I, LDL was prelabeled with [1,2-H]cholesterol ([H]cholesterol) (DuPont NEN) using 10 µCi/100 µg LDL protein. Briefly, 10 µCi of [H]cholesterol solution in toluene was dried to completion under Nonto the wall of a glass tube. LDL protein (100 µg) diluted to 500 ml with 1% bovine serum albumin in Tris-buffered saline-EDTA was added into the [H]cholesterol-coated tube and incubated at 37 °C for 30 min. The incubation mixture was dialyzed against 20 m M Tris buffer, pH 7.7, containing 150 m M NaCl (TBS) in which all cholesterol transfer assays were carried out. The procedure to label LpA-I with [H]cholesterol is essentially the same as above. To monitor the POPC transfer between lipoproteins, the donor particles were incubated with 10 µCi of [ oleoyl-1-C]POPC ([C]POPC) (DuPont NEN) coated on the glass wall as described above. To monitor the potential apoA-I movement during the process of cholesterol transfer assay, apoA-I in LpA-I were iodinated by Iodo-Bead technique (Pierce).

Monoclonal Antibodies

Antibodies 4H1, 3G10, and 5F6 were prepared in our laboratory (25) . Antibodies 2G11 and 4A12, characterized by Petit et al. (26) , were purchased from Sanofi (Paris, France). Antibodies A05, AO3, A44, and r5G9 are a generous gift of Dr. Fruchart (SERLIA, Institut Pasteur, Lille). The apoA-I sequence forming the epitopes for each of these mAbs is indicated in and was previously delineated in our lab by their immunoreaction to synthetic peptides, -galactosidase fusion proteins and CNBr fragments of apoA-I molecules (15) . Antibody 2H2 against rat synthetic atrial natriuretic factor and Bsol.12 against solubilized apo B (27) were used as control. All of the mAbs used are murine IgG, which were purified on protein G- or protein A-Sepharose columns (Pharmacia). Fab fragments of these mAbs were prepared by papain digestion and purified by protein A-Sepharose affinity chromatography.

Cholesterol Transfer Assay between LDL and LpA-I Particles

All components for this assay were dialyzed against 4 liters of TBS at 4 °C overnight immediately prior to the experiment. LpA-I particles diluted in 100 µl of 1% bovine serum albumin-TBS were preincubated at 37 °C for 30 min with serial dilution of mAbs in 200 µl of TBS, which gave mAb/apoA-I molar ratio between 2 and 0.0625. LDL diluted in 160 µl of 1% bovine serum albumin-TBS was added to initiate the cholesterol transfer process. The mixtures were incubated at room temperature for 20 min. The transfer reactions were stopped in an ice bath. Heparin-Mnprecipitation procedure was employed to separate LDL and LpA-I as described by others (6, 10) . Briefly, 25 µg of carrier LDL in 50 µl of Tris buffer, 100 µl of 0.14% heparin, and 25 µl of 1 M MnClwere added into each tube. The tubes were vortexed, kept on ice for 10 min and then centrifuged in a Microfuge (Eppendorf, Westbury, NY) at 15,000 rpm for 10 min. The radioactivities of precipitates containing mainly LDL and the supernatants containing LpA-I were subsequently counted in a scintillation counter. A correction factor was included in the calculations to correct for the constant amount of LpA-I, which is co-precipitated and is independent of the presence of antibodies.

For cholesterol transfer from LDL to LpA-I, LDL was labeled with [H]cholesterol, and the assays were done at LpA-I/LDL particle ratio of 40:1. A typical incubation mixture included 2.1 µg of LpA-I protein, 0.5 µg of LDL protein, and mAb starting at 22.5 µg/tube with 1:2 serial dilution. For cholesterol transfer from LpA-I to LDL, LpA-I was labeled with [H]cholesterol, and the assays were done at LpA-I/LDL particle ratios of 2:1. Particle ratio is calculated using the molar apolipoprotein composition. A typical incubation mixture includes 1.0 µg of LpA-I protein, 4.5 µg of LDL protein, and mAb starting at 12 µg/tube with 1:2 serial dilution. These conditions were determined and chosen within linear range of cholesterol transfer according to the kinetic studies described elsewhere (28) . No significant difference in the effects of the mAbs tested were observed during incubations varying from 20 to 60 min.

Cholesterol transfer rate as a function of incubation time was calculated essentially as described by others (10) . The negative and positive values represent inhibitory and enhancement effects of the mAbs on cholesterol transfer, respectively.

Measurement of Cholesterol Mass Transfer from LDL to LpA-I in the presence of Anti-apoA-I Antibodies

The transfer reactions were done under the same conditions as above but without isotope labeling and scaled up 10-fold in order to obtain sufficient material for enzymatic assay of cholesterol mass. After incubation for transfer and precipitation of LDL by heparin-Mn, the supernatants (LpA-I compartment) were concentrated 5 times by speedvac concentrator (Savant, Farmingdale, NY), and cholesterol mass in these supernatants was determined by enzymatic kits (Boehringer Mannheim).

Separation of Lp2A-I (9.6 nm) or Lp2A-ImAb Complexes and LDL by Agarose Gel Electrophoresis

LpA-I labeled with [4-C]cholesterol ([C]cholesterol) were incubated with appropriate mAbs in the presence or absence of LDL at the same ratio as for the transfer assay but in smaller volumes. The reaction mixtures were applied onto Paragon Lipo agarose gel electrophoresis system (Beckman, Brea, CA) and run at 100 V for 30 min. The gels were fixed with 10% acetic acid for 5 min and baked to dryness at 80 °C. The dry gels were either autoradiographed or sliced into 2-mm fractions for counting of radioactivity. Pure IgG preparation of some mAbs were also iodinated and run in agarose gel electrophoresis to compare their electrophoretic mobility with that of their immunecomplexes.

Separation of Lp2A-I or Lp2A-ImAb Complexes from LDL by Native Gradient Gel Electrophoresis

The Lp2A-I, mAbs, and LDL were incubated as described for cholesterol transfer assay. At the end of incubation, the mixtures were immediately applied onto precast native gradient gel (4-20%) (Bio-Rad). The electrophoresis were run at 100 V at room temperature for 6 h, and the gel was stained with Coomassie Brilliant Blue. In studies to exclude the possibility of fusion between LpA-I and LDL particles during the cholesterol transfer assay, Lp2A-I, -3A-I, and -4A-I were labeled with I, incubated with LDL as in the transfer assay, and then separated by native gradient gel electrophoresis. The distribution of radioactivity was then assessed by autoradiography.

Circular Dichroism of Immunecomplexes of LpA-I Particles

The effect of anti-apoA-I mAbs on the secondary structures of apoA-I molecules in the LpA-I/anti-apoA-I immunecomplexes were studied by subtractive CD on a Jasco J-40A spectropolarimeter. All samples and mAbs were dialyzed against 0.5 m M phosphate buffer, pH 7.4, at 4 °C overnight. LpA-I particles were preincubated with different mAbs at mAb/apoA-I molar ratio of 1 in duplicates. Molar ellipticities were read at 222 nm at 24 °C in a 0.1-cm path length quartz cell. Percent -helix of the LpA-I or their immunecomplexes were calculated from the molar ellipticities at 222 nm (29) using a mean residue weight for apoA-I of 115.3 and for mouse IgG of 112.5.


RESULTS

Effects of Anti-apoA-I mAbs on Cholesterol Transfer between LDL and Lp2A-I

Discoidal LpA-I prepared in the presence of cholate are well characterized. Variations in the phospholipid, cholesterol, and apoA-I molar ratios generates distinct populations of homogeneous particles with different lipid to apoA-I ratios or with different numbers of apoA-I/particle (18, 19, 20) . We first studied Lp2A-I (9.6 nm), which represents the major and most stable particle amongst the Lp2A-I species (18, 19, 20) . The acceptor Lp2A-I (9.6 nm) particles were preincubated with serial dilutions of anti-apoA-I mAbs or control mAbs, and then [H]cholesterol-labeled LDL were added at Lp2A-I/LDL particle molar ratio of 40:1 to evaluate the effects of anti-apoA-I mAbs in the cholesterol transfer from LDL to Lp2A-I. The apoA-I sequences attributed to each epitope are indicated in . The results are summarized in Fig. 1and . Antibodies 2H2 and Bsol.12 are nonrelevant mAbs used as negative controls. The following are antibodies that strongly inhibited cholesterol transfer from LDL to Lp2A-I, with the position of their epitopes indicated in the parenthesis: A05 (residues 25-82), 2G11 (residues 25-110), A03 (residues 135-140), r5G9 (residues 149-186), A44 (residues 149-186), and 4A12 (residues 173-205). The inhibitory effects start at mAb/apoA-I molar ratio of 0.25 and reach saturation at mAb/apoA-I molar ratio between 0.5 and 1. However, mAbs 4H1 (residues 2-8) and 3G10 (residues 96-121) do not have any significant effect on cholesterol transfer from LDL to Lp2A-I (9.6 nm) in comparison with the control mAbs. Antibody 5F6 (residues 116-141) marginally increases the cholesterol transfer (20%) compared with the variation in controls (10-15%), but this effect is not mAb dose-dependent. The affinity of these mAbs for Lp2A-I (9.6 nm) particles were previously determined by immunoprecipitation using Pansorbin armed with rabbit anti-mouse IgG and I-labeled LpA-I (30, 31) . Using these parameters, no correlation is seen between the affinity of these mAbs for LpA-I and their effects on cholesterol transfer.


Figure 1: Effects of anti-apoA-I monoclonal antibodies on unesterified cholesterol transfer from LDL to Lp2A-I (9.6 nm). Lp2A-I (9.6 nm) was preincubated with mAbs at mAb/apoA-I molar ratios between 0.125 to 2, and then LDL was added at Lp2A-I/LDL particles ratio of 40:1 to initiate the cholesterol transfer process. A typical incubation mixture included 2.1 µg of LpA-I protein, 0.5 µg of LDL protein, and mAb starting at 22.5 µg/tube with 1:2 serial dilution. The data are means calculated from three independent assays in duplicate. Anti-apoA-I mAbs tested are 4H1 (), 2G11 (), A05 (), 3G10 (▾), 5F6 (), A03 (), A44 (), r5G9 (), and 4A12 (). Antibodies Bsol.12 () and 2H2 () are nonrelevant mAbs used as negative control.



The effects of Fab fragments of 3G10, A03, A44, and 4A12 on cholesterol transfer from LDL to Lp2A-I were also tested. The inhibitory effects of A03, A44, and 4A12 remain but are slightly attenuated (between 60 and 80% at Fab/apoA-I molar ratio of 4) (Fig. 2). Fabs of 4H1 and 5F6 did not affect cholesterol transfer significantly (data not shown).


Figure 2: Effects of Fab fragments of anti-apoA-I mAbs on cholesterol transfer from LDL to Lp2A-I (9.6 nm). Fab fragments were prepared from IgG by papain digestion and purified by affinity chromatography on a protein A-Sepharose column. Purity of these Fab fragments were proven by SDS-polyacrylamide gel electrophoresis. Fabs 3G10 (▾), A03 (), A44 (), and 4A12 () retain their specific binding activities to apoA-I as tested by RIA. Fab 2H2 () is a negative control. Data are calculated from two repeated assays.



When [H]cholesterol-labeled Lp2A-I (9.6 nm) was used as cholesterol donor to test the influence of mAbs on cholesterol transfer from Lp2A-I (9.6 nm) to LDL, all of the mAbs that inhibit cholesterol transfer from LDL to LpA-I do enhance cholesterol transfer from this Lp2A-I to LDL (). The only difference is observed with mAb 3G10, which does not significantly affect cholesterol transfer from LDL to Lp2A-I, but increases the cholesterol transfer from Lp2A-I (9.6 nm) to LDL by about 70%.

Since specific anti-apoA-I mAbs both stimulate the transfer of cholesterol from Lp2A-I to LDL and inhibit transfer in the opposite direction, net mass transfer of cholesterol from LDL to LpA-I must take place. Indeed cholesterol mass in the supernatant fractions (LpA-I compartment) is significantly reduced after incubation with LDL in the presence of mAbs such as A05, A03, A44, and 4A12, but not with 4H1 or control mAb 2H2 (Fig. 3).


Figure 3: Effects of mAbs on cholesterol mass in Lp2A-I. After incubation with LDL, the cholesterol transfer assays were done at Lp2A-I/LDL particle ratio of 40:1 and mAb/AI molar ratio of 1, but scaled up to 10-fold compared with the ordinary assay. Cholesterol mass remaining in the Lp2A-I after the transfer assay by heparin-Mnprecipitation was measured; data represent means of two independent assays in quadruplicate. Error bars indicate ± S.D. In comparison to mAb 2H2, the presence of mAbs A05, A03, A44, or 4A12 significantly reduced the cholesterol concentration in the Lp2A-I compartment.



To address the possibility of artifacts related to the binding of mAbs to heparin or LDL, we have tested the binding of the anti-apoA-I mAbs to heparin or LDL by radioimmunoassay. The removawells were coated with heparin-Mnreagent or LDL and incubated with these mAbs and then with iodinated anti-mouse IgG. None of these mAbs showed binding affinity for heparin or LDL (data not illustrated).

The effects of these anti-apoA-I mAbs on cholesterol transfer to and from LDL were also studied using other Lp2A-I particles of different sizes. Very similar results were observed, and the same mAbs inhibited transfer from LDL to Lp2A-I and enhanced transfer in the opposite direction (). In contrast, these antibodies have no effect on the transfers to and from Lp2A-I (7.8 nm) (not illustrated), the smallest and most cholesterol enriched particle in the Lp2A-I class. This particle also is unstable and tends to release free apoA-I from which it is difficult to separate. We attribute the lack of antibody effect on transfer reactions with this particle to its extreme composition and its unstability.

We have also observed no effect by anti-apoA-I mAbs on cholesterol transfer between LDL and either Lp3A-I or Lp4A-I (not illustrated). The kinetics of these transfer have been studied in details elsewhere (28) . Lp3A-I or -4A-I of a size and composition identical to these studied here were found to exchange very little cholesterol with LDL (28) , whereas Lp2A-I was the most active donor and acceptor. We attribute the lack of the effect of the mAbs on cholesterol transfer to the large particles to the slow transfer rates.

Effects of mAbs on the Transfer of apoA-I and Phospholipids from LpA-I to LDL

The effect of mAbs on the transfer of lipoprotein components other than cholesterol between Lp2A-I and LDL were also evaluated. Lp2A-I (9.6 nm) particles labeled with either I-labeled apoA-I or [C]POPC were incubated with mAbs and LDL under conditions similar to those used for the study of cholesterol transfer and separated from LDL by heparin-Mn. There is no significant correlation between the transfers of either I-labeled apoA-I or [C]POPC of Lp2A-I (9.6 nm) to LDL and cholesterol transfers between the same particles. This demonstrates that LpA-I is not transferred or precipitated as a whole particle, but that different mAbs appear to have specific effects on the transfer of cholesterol and phospholipids ().

Only antibody A44 causes a significant increase in the precipitation of I-labeled LpA-I (17%) together with the increased transfer of cholesterol (100%) and phospholipids (18%). Most mAbs that enhance transfer of cholesterol from LpA-I to LDL, also significantly enhance phospholipid transfer (2G11, A05, 3G10, A03, A44), whereas those that have minimal or no effect on cholesterol transfer have no effect on phospholipids (4H1, 5F6). The exceptions are 4A12, which elicits a highly significant transfer of cholesterol but not of POPC and apoA-I, and 3G10, which has a weak effect on cholesterol transfer compared with its effect on POPC and apoA-I transfer.

Electrophoretic Migration of Lp2A-I after Incubation with mAbs in the Presence or Absence of LDL

To exclude the possibility that in the presence of LDL, the binding of mAbs to LpA-I may induce the destabilization or rearrangement of Lp2A-I and thereby nonspecific incorporation of Lp2A-I constituents into LDL (fusion of lipoprotein particles), I-labeled Lp2A-I (9.6 nm) were prepared and incubated with mAbs in the presence and absence of LDL. The migration profiles of Lp2A-I particles on agarose gel electrophoresis are affected differently by the presence of specific mAbs, and these shifts in migration provide evidence for the binding of mAbs to Lp2A-I particles. However, the presence or absence of LDL does not induce any additional change in migration LpA-I or LpA-ImAb complexes (Fig. 4, A-C). Therefore, the difference of mobility of the immunecomplexes reflects the difference in surface charge of each mAb (Fig. 4 D).


Figure 4: Agarose electrophoretic migration profile of mAb-Lp2A-I (9.6 nm) complexes in the presence and absence of LDL. Panels A, B, and C, anti-apoA-I mAbs were incubated with I-labeled Lp2A-I (9.6 nm) in the presence or absence of LDL under the optimal conditions determined for the cholesterol transfer assay, i.e. 2 µg of I-labeled Lp2A-I protein (20,000 cpm), 11.3 µg of mAb, and with or without 0.5 µg of LDL. At the end of incubation, aliquots of the mixtures were immediately loaded on agarose gel and run at 100 V for 30 min. The gels were fixed with 10% acetic acid, baked to dryness, and then subjected to autoradiography. Antibodies 2H2 and Bsol.12 are negative control. Panel D, agarose gel electrophoretic mobility of iodinated anti-apoA-I mAbs. All of these mAbs were iodinated by IODO bead technique (Pierce). The samples were loaded at 100,000 cpm/lane and run at the same condition as that for lipoproteins.



The same incubation mixtures were also applied onto native gradient gel electrophoresis. As shown in Fig. 5, LDL do not enter the separation gel in a 4-20% gradient gel. In the incubation mixture containing phosphate-buffered saline or control mAb 2H2, Lp2A-I (9.6 nm) migrate to their normal position, while in the presence of mAbs 4H1, 2G11, A03, 5F6, 3G10, A44, and 4A12, Lp2A-I formed complexes of different sizes as indicated by the migration bands with much higher molecular weights. Immunoblots using anti-mouse IgG and anti-apoAI showed that these high molecular weight bands are immunecomplexes rather than larger LpA-I particles (data not shown). As mentioned above, when I-labeled LpA-I are used in the same experiments, there is no radioactivity incorporated into LDL zone (data not illustrated). This demonstrates that there is no particle fusion of LpA-I with LDL during incubation for cholesterol transfer.


Figure 5: Native gradient gel electrophoresis migration pattern of Lp2A-I (9.6 nm) antibody complexes in the presence of LDL. Anti-apoA-I mAbs were incubated with Lp2A-I (9.6 nm) in the presence of LDL under the optimal condition as the cholesterol transfer assay, i.e. 2 µg of Lp2A-I protein, 11 µg of mAb, and 0.5 µg of LDL protein. Antibody 2H2 is a negative control. At the end of incubation, the mixtures were loaded and run in native 4-20% gradient polyacrylamide gel and run at 100 V for 6 h. The protein migration bands were visualized with Coomassie Brilliant Blue staining.



Evaluation of Cholesterol Transfer by Agarose Gel Electrophoresis

To assess cholesterol transfer by another assay, we have analyzed by agarose gel electrophoresis the movement of [C]cholesterol from Lp2A-I (9.6 nm) bands to LDL bands after incubation in the presence of mAbs. However, the electrophoretic mobility of Lp2A-I (9.6 nm) complexed with mAbs 4H1, 3G10, A44, and 4A12 overlaps more or less with that of LDL (Fig. 4), limiting the application of this method to the effect of mAbs A03, 2G11, and A05 on transfer. In these experiments, [C]cholesterol-labeled Lp2A-I were incubated with the mAbs and LDL, and the incubation mixture was separated by agarose gel electrophoresis, and the [C]cholesterol radioactivity present in the LpA-I and LDL fractions was measured. Compared with phosphate-buffered saline and control mAb 2H2, A03, 2G11, and A05, increased cholesterol transfer from Lp2A-I (9.6 nm) to LDL and only the effect of mAb A05 did not reach significance (I).

Secondary Structure of apoA-I in LpA-ImAb Complexes

To understand the nature of the interference of mAbs on cholesterol transfer with Lp2A-I, we assessed the effects of the anti-apoA-I mAbs on the secondary structure of apoA-I in these particles. Lp2A-I (9.6 nm) was incubated with various anti-apoA-I mAbs at an antibody/apoA-I molar ratio of 1, and the -helicity of resulting Lp2A-ImAb complexes, free Lp2A-I particles, and free mAbs was measured by circular dichroic spectroscopy at the wavelength of 222 nm. Nonrelated mAbs against atrial natriuretic peptide or soluble apoB were used as control. Immunoglobulins contain very little -helix secondary structure and studies have shown that binding to an antigen has a negligible effect on the antibody helical structure (32) . Therefore, the ellipticity values of each mAb were measured and used as background to correct the CD values of each corresponding immunecomplexes. In our study, the -helicity contents of free Lp2A-I (9.6 nm) is 69.2%, in agreement with earlier studies by Jonas et al. (33) . Compared with the mean of control values obtained with irrelevant mAbs preincubated with Lp2A-I (9.6 nm), the -helicity of the immunecomplexes formed with most anti-apoA-I mAbs is significantly increased (). This suggests that the binding of antibodies to Lp2A-I does not destablilize apoA-I but, on the contrary, may increase the stability of amphipathic -helices. However, there is no correlation between the capacities of mAbs to increase the -helicity in the Lp2A-I complex and to interfere in cholesterol transfers involving these particles.


DISCUSSION

The observation that specific anti-apoA-I antibodies inhibit cholesterol transfer from LDL to LpA-I and enhance transfer in the opposite direction, resulting in a net loss of cholesterol from LpA-I, was unexpected and incompatible with the initial hypothesis that formed the basis of our experimental approach. Indeed such effects cannot be related to a simple steric hindrance but must also imply specific conformational modifications of the Lp2A-I particle that modify cholesterol binding.

To rule out any possibility of artifact, the cholesterol transfer assay has been extensively verified and validated, especially when carried out in the presence of antibodies. The antibodies have been shown not to interact with the heparin-Mnsystem. Also, the analysis of the lipoproteins incubated in the presence of the different mAbs demonstrates the quantitative binding of the antibodies to all LpA-I particles and provides no evidence for the fusion of LpA-I and LDL in the presence of mAbs as judged by agarose gel electrophoresis and no evidence of dissociation of Lp2A-I and generation of lipid-free apoA-I as judged by gradient gel electrophoresis. In addition to the demonstration of the stability of LpA-I immunecomplexes in the transfer assay and to the absence of LDL-LpA-I fusion, it was important to also show that similar results could be obtained with a totally different assay, such as by separation of LDL and LpA-I by agarose gel electrophoresis.

Both cholesterol and phospholipids have been proposed to transfer by collision- or diffusion-mediated mechanisms (6, 7) , and simultaneous transfers of these lipids have been demonstrated from cells to apolipoproteins (34) . This suggested to us that mAbs could have simultaneous effects on the transfer of both lipids. However, when we analyzed the effects of mAbs on the transfer of other components of LpA-I, phospholipids, and apoA-I, the picture of transfer that emerges is one that appears to vary between mAbs and affect each component individually. Among all antibodies tested, only A44 enhances transfer of I-labeled apoA-I to LDL. Although this enhanced transfer represents only 10% above background, it is nevertheless significant and suggests that A44 may cause the association or coprecipitation with LDL of about 1 in 10 apoA-I molecules. All but one mAb, which enhances cholesterol transfer from Lp2A-I to LDL by about 70% or more, also significantly increases phospholipid transfer by 14-22%. The exception is 4A12, which although is the strongest stimulator of cholesterol transfer (150%) elicits no change in phospholipid transfer. The two mAbs that have no significant effect on cholesterol transfer, 4H1 and 5F6, also have no effect on phospholipid transfer. For those mAbs, which stimulate cholesterol and phospholipid transfers from Lp2A-I to LDL, we can calculate from the initial lipid concentrations in Lp2A-I and from the transfer rates () that under basal conditions and in the absence of mAbs, 0.76 nmol of cholesterol and 0.23 nmol of phospholipid transfer per hour from Lp2A-I to LDL, a ratio of about three moles to one; typical stimulatory mAbs increase transfer to 1.5 mol of cholesterol and 0.26 nmol of phospholipid or a ratio of 6 to 1 mol. The ratio of cholesterol to phospholipid transferred measured here is comparable with that of 6 mol to 1 between unilamellar donor vesicles and neutral acceptor vesicles reported by McLean and Phillips (6) . The net effect of the antibody-mediated stimulation is therefore to enhance the transfer of cholesterol relative to phospholipids.

As shown by the ellipticity of the immunecomplexes, most antibodies increase the -helicity of apoA-I, suggesting that they may stabilize the LpA-I. We hypothesize that depending on the position of epitopes, i.e. specifically for those in amphipathic -helices, specific anti-apoA-I may increase the binding of apoA-I to phospholipid, which would increase the competition of the amphipathic domain with cholesterol and thus promote cholesterol desorption and transfer to other lipoproteins. This would be compatible with the concept proposed earlier by others that apoA-I compete with cholesterol for solvation in or binding to the phospholipid phase (35) . The absence of correlation between mAb effects on -helicity and lipid transfers should not be unexpected since apoA-I structure is complex, and different domains are predicted to have different interactions with lipids. As discussed below, mAb 4H1 reacts with an epitope that is close to the central domain. While 4H1 epitope does not contain any predicted -helix, this antibody enhances -helicity, and we propose that it does exert this effect indirectly through its overlap with other regions predicted to have significant -helicity, such as that for 5F6.

The difference between antibodies in eliciting either simultaneous or individual transfer of cholesterol and phospholipids suggests that the distribution of these lipids in relation to apoA-I could vary between domains or that different domains compete differently for binding to the phospholipids. Calorimetric and solubility studies of reconstituted HDL with varying cholesterol have shown that cholesterol is excluded from about 55% of the phospholipid molecules bound to the apolipoprotein in a nonmelting state, which constitutes the phospholipid boundary layer (36) . Incorporation of fluorescent labeled cholesterol and phospholipids in reconstituted HDL has also demonstrated that the fraction of cholesterol adjacent to apoA-I is less than that of phospholipids (35) , and this has been interpreted as the result of a competition between apoA-I and cholesterol for hydrophobic solvation by phospholipids. This concept is supported by measurements of interfacial tension of phosphatidylcholine and cholesterol monolayers that exhibit a strong cooperative lateral interaction between the two lipids. In the presence of apoA-I, this cooperation is replaced by a strong positive interaction between apoA-I and phospholipids and a weak lateral interaction between apoA-I and cholesterol (37) . These observations could also be compatible with the notion that different pools of cholesterol may exist within lipoprotein particles and that their distribution may also vary in relation to apoA-I domains. This would explain the differential effects of mAbs on cholesterol and phospholipid transfers, and mAbs against different regions of apoA-I could affect cholesterol transfer through different mechanisms.

The specificity of the inhibitory mAbs is made more significant by the existence of other mAbs that have no effect on cholesterol transfers between LDL and LpA-I, and it is interesting to consider the location of their epitopes in relation to what is known of apoA-I structure. The epitopes of the neutral mAbs are located in two distinct sequences, 4H1 at the extreme N terminus, residues 2-8, and 3G10 and 5F6 in a central domain centered around residue 121 and spanning residues 99-135 (Table I and Fig. 6). These two domains have been already identified as being closely related, both structurally and functionally. Our initial competition assays have shown that the N-terminal domain (identified by mAb A05) is close to the central domain (21) , but 4H1, which was then used as capture antibody, could not be entered in these competition assays. More recent competition experiments using the capture antibody as a competitor have brought further evidence showing that these two domains, including 4H1, are indeed very close in discoidal LpA-I (Fig. 6). However it should be noted that such composite domain can be intermolecular rather than intramolecular as depicted here, i.e. involving the N terminus of one apoA-I and the middle domain of another. These antibodies also have similar effects on the cholesterol esterification reaction (31) ; 4H1 and 5F6 both enhance lecithin:cholesterol acyltransferase reaction with small Lp2A-I (7.8 nm), while 4H1, 2G11, and 5F6, enhance lecithin:cholesterol acyltransferase reaction with small Lp3A-I (10.8 nm). It is therefore reasonable to conclude that the epitopes for 4H1 and 5F6 are located within the same domain on apoA-I three-dimensional structure and also share the same functional specificity, including a similar lack of effect of mAbs binding to these sites on cholesterol transfer. This neutral domain separates two lipid binding domains, residues 25-110 or 25-120 and residues 135-205, which, based on the present results, are those competing with cholesterol for binding to phospholipids. Thus the binding of mAbs to epitopes located in these domains decreases cholesterol retention by LpA-I by increasing apoA-I interaction with phospholipids.


Figure 6: Model of apoA-I structure on discoidal LpA-I and location of epitopes recognized by anti-apoA-I mAbs inhibitory or enhancing for cholesterol transfer between LDL and Lp2A-I (9.6 nm). The different amphipathic -helices (represented by rectangles) run parallel to the axis of the phospholipid disc, are antiparallel to each other, and linked by coiled regions and -turns (represented by a string). The areas identifying the epitopes are represented by different shades of gray beneath the motifs of secondary structure. The epitopes for mAbs without effect on cholesterol transfers are represented in areas of darkest gray.



Our conclusions are in general compatible with recent results of others on the effects of specific mAbs on cellular cholesterol efflux, which we also interpret as defining a neutral central domain separating two domains where the binding of mAbs influence cholesterol transfer. Luchoomun and co-workers (38) identified two mAbs reacting at residues 25-82 (A05) and at residues 149-186 (A44) that do inhibit cellular cholesterol transfer to LpA-I, while mAbs reacting between residues 99-132 and 135-140 do not. Fielding et al. (39) have described two mAbs reacting between residues 137-144 and between residues 113-128 . . . 141-148, the later being a discontinuous epitope that inhibits cellular cholesterol transfer to pre-LpA-I while mAbs reacting between 93-99 and 167-174 do not. Banka et al. (40) have also observed inhibition of efflux by mAbs reacting between residues 95-110 and 74-105. While there is a general concordance in the location of epitopes for the mAbs inhibitory to cholesterol transfers, there is also uncertainty for the exact boundaries of these domains. Considering the variation that we observed ourselves with different Lp2A-I (Tables I and II), we attribute the variation to the different conformations that apoA-I assumes in these different lipoproteins. This is particularly true for the different lipoproteins used in the studies cited above. Luchoomun and colleagues (38) use discoidal LpA-I; Fielding et al. (39) studied the partially defined plasma pre-LpA-I; and Banka et al. (40) used HDL and apoA-I proteoliposomes.

In conclusion, although we need more information for a complete interpretation of the role of apoA-I domains in cholesterol transport and metabolism, we have presented evidence for the direct link between apoA-I structure and cholesterol transfers and for the existence of apoA-I domains that differ in their interaction with cholesterol versus phospholipids. Furthermore we can make several hypothesis for further testing. It is clear that as initially suggested by others (14) , the interface between apoA-I and lipids on the edge of discs introduce incongruities in lipid packing. We have further defined this concept and provided evidence that cholesterol binding is linked to defined regions of apoA-I and depends on the conformation of specific domains. The present report suggests that cholesterol association and desorbtion from LpA-I may involve particular domains because mAbs binding to epitopes overlapping specific amphipathic -helices enhance cholesterol transfer. Finally, the effects of specific mAbs on the binding of cholesterol to LpA-I and on its desorption from LpA-I appears to demonstrate that the cholesterol content of lipoprotein particles is regulated by the apolipoprotein conformation.

  
Table: Effects of antibodies on the transfer of Lp2A-I (9.6 nm) constituents to LDL


  
Table: Effects of anti-apoA-I monoclonal antibodies on cholesterol transfer between LDL and Lp2A-I (8.6 nm and 10.2 nm)


  
Table: Effect of antibodies on cholesterol transfer from Lp2A-I (9.6 nm) to LDL measured by agarose gel electrophoresis

Lp2A-I (9.6 nm) labeled with [C]cholesterol were preincubated with mAbs at mAb/apoA-I molar ratio of 2 at 37 °C for 30 min, and LDL were added and incubated at 25 °C for another 20 min. LDL and Lp2A-I in incubation mixtures were separated by agarose gel electrophoresis. Each lane of the gel were sliced into 2-mm fractions and counted for -radioactivity. Data represent mean ± S.E. calculated from two experiments.


  
Table: Effects of anti-apoA-I monoclonal antibodies on predicted secondary structure of apoA-I in Lp2A-I (9.6 nm) immunecomplex



FOOTNOTES

*
This work was supported by a grant from the Medical Research Council of Canada (PG-27). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Medical Research Council fellowship. Present address: Service de biochimie médicale, L'Hotel-Dieu de Québec 11, Côte du Palais, Québec G1R 2J6.

To whom correspondence should be addressed: H460, University of Ottawa Heart Institute, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9. Tel.: 613-761-5254; Fax: 613-761-5281.

The abbreviations used are: LpA-I, lipoprotein containing apoA-I; mAb, monoclonal antibody; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; LDL, low density lipoprotein; HDL, high density lipoprotein.


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