THE JOURNAL OF Bro~oorc~~ CHEMISTRY Vol. 254, No. 7, Issue of April 10, pp. 25173525, 1979 Printed in U.S.A.

Apolipoprotein A-II and Structure of Human Serum High Density Lipoproteins AN APPROACH BY REASSEMBLY TECHNIQUES*

(Received for publication, May 17, 1978, and in revised form, August 28, 1978)

Mary C. RitterS and Angelo M. Scanu

From the Departments of Medicine and Biochemistry, The University of Chicago Pritzker School of Medicine, and The Franklin McLean Memorial Research Institute,8 Chicago, Illinois 60637

In an effort to determine the structural role of human serum apolipoprotein A-II (apo-A-II) in high density lipoproteins (HDL), we have conducted detailed studies on the conditions that influence the in vitro complexa- tion of apo-A-II with mixtures of the polar and nonpolar lipids obtained from the parent HDL. APO-A-II and HDL lipids in different stoichiometric amounts were co-sonicated under different medium conditions. The resulting complexes were separated from the unreacted products by ultracentrifugal techniques, gel permea- tion chromatography, or both, and then characterized by physicochemical means. As defined by ultracen- trifugal criteria, a series of HDL complexes were formed. They were heterogeneous in size (90 to 200 A) and hydrated density (1.20 to 1.063 g/ml), although they had general properties of native HDL by electro- phoretic and chromatographic criteria. Recoveries of these complexes were maximal when apo-A-II, at an initial concentration of 11 PM, was reacted with the same weight of lipids. The composition and yield of the complexes were independent of pH (pH 6.6 to 8.6) and ionic strength (10 to 60 111~). At low ionic strength (2 mu), however, less lipid associated with apo-A-II. At apoprotein concentrations below 11 PM (lower limit, 0.2 PM), the amount of reassembled HDL was minimal. Under these conditions, the lipid-free apo-A-II ex- hibited a marked decrease in molar ellipticity. At higher protein concentrations (upper limit, 108 PM),

recovery of the reassembled HDL was again minimal; at these levels, the apoprotein demonstrated an in- crease in molar ellipticity, probably related to self-as- sociation. At lipid/protein weight ratios above 1, apo- A-II was mainly recovered in a class of lighter lipid- rich particles floating at density 1.063 g/ml; this class became more predominant as the initial lipid/protein ratio was increased. The lipid-rich particles were larger (150 to 540 A) than the reassembled HDL and were heterogeneous in hydrated density (1.050 to 1.023 g/ ml); their lipid composition resembled that of multila- mellar lipid vesicles which had been isolated from son- icated mixtures of HDL lipids in the absence of apo-A-

* This research was supported in part by funds from the United States Public Health Service Grant HL-18577. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Chicago and Illinois Heart Association Research Fellowship F72-3 and United States Public Health Service Postdoc- toral Fellowship HL-53,817.

§ Operated by The University of Chicago for the United States Energy Research and Development Administration under Contract EY-76-C-02-0069.

II. However, they differed in hydrated density, size, shape, and particle weight from that of the lipid vesi- cles.

The results indicate that (a) apo-A-II can generate HDL complexes, although these are heterogeneous in size and density; (b) the formation of these complexes requires well defined conditions, particularly with re- gard to initial protein concentration and lipid/protein ratio; (c) below and above the optimal apo-A-II concen- tration for HDL reassembly, reassociation of apo-A-II with lipids is minimal, probably due to changes in conformation or state of association, or both, of the apoprotein; and (d) at lipid/ape-A-II ratios above 1, the formation of lipid-rich particles is due to a process characterized by an adsorption of apo-A-II to thermo- dynamically stable lipid vesicles followed by their dis- ruption into particles heterogeneous in size and den- sity. We conclude that the interaction of apo-A-II with HDL lipids is extremely sensitive to experimental con- ditions and that apo-A-II can be incorporated into lip- oprotein particles of much broader distribution than those previously obtained in similar reassembly studies involving apolipoprotein A-I (Ritter, M. C., and Scanu, A. M. (1977) J. BioL Chem. 252,1208-1216).

Apolipoprotein A-II, one of the major proteins of the human serum high density lipoprotein class, has a molecular weight of 17,400 and is composed of two identical polypeptide chains linked together by a single disulfide bridge (l-3). Previous work in this and other laboratories (4-6) has shown that apo- A-II’ self-associates in aqueous solutions and that it exhibits changes in molecular properties as a function of protein con- centration and nature of the aqueous medium. A comprehen- sive review has recently appeared on this subject (7). The lipid binding of apo-A-II has been examined in a number of laboratories (8-15); however, the influence of initial protein concentration and medium conditions on this process was not assessed. The role played by apo-A-II in HDL structure is still unknown. In order to provide information on the subject, we have systematically investigated the interaction between hu- man apo-A-II and HDL lipids in u&o. The results obtained have indicated that the interaction of apo-A-II with lipids is highly dependent on the initial concentrations of the reactants and on their stoichiometry and that the resulting complexes

’ The abbreviations used are: APO-A-II, apolipoprotein A-II; apo- A-I. aoolinonrotein A-I: ano-HDL. aoonrotein of HDL: HDL. hieh density lipoprotein (density, 1.063 tb i.21 g/ml); H~LJ, Hl!lL ‘bf density 1.125 to 1.21 g/ml; R-HDL (apo-A-II), reconstituted HDL containing apo-A-II.

2517

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2518 Interaction of APO-A-II with HDL Lipids

have a broad density and size distribution. A preliminary report on these findings has been published (16).

MATERIALS AND METHODS

Isolation of APO-A-II from HDL

Human serum HDL was isolated from the blood of normal, healthy, fasting male donors (A+) by ultracentrifugal flotation, as previously described (17). The HDL preparations were dialyzed extensively against a solution of 0.15 M NaCl and 0.05%> EDTA, pH 7.0, and delioidated at -10°C with 3:2 (v/v) ethanol/ethvl ether (18). The apoprotein of HDL was fractionated by Sephadex G-200 gel permea- tion chromatography in 8 M urea (19), followed by further purification of Peak IV by ion exchange column chromatography on DEAE- cellulose (Whatman DE52 microgranular) in 8 M urea (20). The apo- A-II thus obtained migrated as a single band during polyacrylamide gel electrophoresis in 8 M urea (21) and in 0.1% sodium dodecyl sulfate (22). Whenever checked, it had the same amino acid composition published previously (3).

Isolation of HDL Lipids

The lipids obtained by ethanol/ether extraction of HDL (18) were concentrated by flash evaporation and stored in 9:l (v/v) chloroform/ methanol under nitrogen at -15°C. The weight per cent composition of the HDL lipids was: phospholipid, 59; cholesteryl ester, 29; free cholesterol, 8; and triglycerides, 4.

Reassembly Techniques

The sonication procedure was identical to that described previously (23). Preliminary studies indicated that sonication of the lipids in a protein-free medium induced no chemical modification of these lipids, as assessed by thin layer chromatography. Sonication of apo-A-II in the absence of lipids under the same conditions as in the reassembly studies caused no detectable changes as determined by amino acid analysis, electrophoretic behavior in polyacrylamide gel electropho- resis in the presence of urea or in 0.1% sodium dodecyl sulfate, immunological response to rabbit anti-human apo-A-II, and circular dichroic measurements. These studies did not exclude the possibility of reversible changes during the sonication process itself. In a typical reassembly experiment, appropriate aliquots of sonicated lipid dis- persions (23) in 0.02 M EDTA, pH 8.6, were incubated at 41’C for 30 min to ensure the gel-liquid crystalline transition of the hydrocarbon chains of the cholesteryl esters (24). Next a solution of apo-A-II, dissolved in 0.02 M EDTA, pH 8.6, was added, and the resulting protein/lipid mixture was co-sonicated under nitrogen (75 watts, three I-min bursts); the average temperature during the sonication process was 50°C. A 30-s ice water cooling period between each sonic burst was used to keep the temperature of the dispersion at about 41°C. The time of sonication was found to ensure complete equilibration between protein and lipid as assessed by the fact that additional co- sonication, regardless of volume, did not influence the results. This was also the case when the time intervals between sonication and fractionation of the products was varied. With this technique, we studied the influence of different conditions on the incorporation of apo-A-II into lipid. protein complexes, namely, apo-A-II concentra- tion, initial lipid/protein ratios, and medium conditions. In the ab- sence of sonication, the simple incubation of a mixture of apo-A-II and HDL lipids at 41°C for 24 h under nitrogen permitted only the formation of an apo-A-II. phospholipid complex. The co-sonication of the same mixture promoted not only interaction of phospholipids with the apoprotein but also incorporation of unesterified and ester- itied cholesterol. These results are in agreement with previous studies utilizing high density apolipoproteins (23-27). For comparative pur- poses in separate experiments, aqueous dispersions of lipids extracted from HDL were subjected to the same sonication procedure used in the reassembly studies. As previously reported (26), native HDL remained unaffected by sonic irradiation.

Separation of Sonicated Mixtures

For some experiments, the lipid/protein mixtures obtained follow- ing co-sonication were separated by ultracentrifugation at a density of 1.006 g/ml, as already reported (23). This step permitted the sedimentation of the unreacted apo-A-II and of any lipid.protein complex formed. In other experiments, the complete separation of the lipids not incorporated into the complex was obtained only by ultra- centrifugation of the lipid/protein mixture at a density of 1.063 g/ml

(23); the undernatant was extensively dialyzed at 8°C against either 0.02 M EDTA, pH 8.6, or 0.15 M NaCl containing 0.01% EDTA, pH 7.0. The undernatants of density 1.006 or 1.063 g/ml were further fractionated by CsCl isopycnic density gradient ultracentrifugation, ultracentrifugal flotation at density 1.21 g/ml, or 8% agarose column chromatography, according to techniques previously described in the reassembly studies with apo-A-I (23). The recoveries of lipid and protein varied from 90 to 100%.

The lipid-rich particles which floated at density 1.063 g/ml were further separated from the unreacted lipids by adjustment of the density to 1.006 g/ml and subsequent centrifugationfor 24 h at 11°C in a 40.3 rotor (Beckman Instruments) at 38,009 rpm. The bottom 2.0 ml contained what we will refer to as lipid-rich particles. These particles were further fractionated by NaCl isopycnic density gradient ultracentrifugation between the limiting densities of 1.063 and 1.018 g/ml for 66 h at 11°C in a SW-40 rotor (Beckman Instruments) at 39,000 rpm or by gel permeation chromatography at 8°C on a silicon- ized column (1.6 x 100 cm) (Pharmacia) of 4% agarose (Sepharose 4B, Pharmacia) at an upward flow rate of 5 ml/h. Other conditions were as previously described (23).

The fractionation of the sonicated HDL lipids was accomplished by NaCl isopycnic density gradient ultracentrifugation and thereafter by Sepharose 4B chromatography as outlined above.

Protein and Lipid Assays

Protein Determination-Protein determinations were performed by a modification (23) of the method of Lowry et al. (28), with bovine serum albumin (Miles Laboratories) used as the standard. For the detection of proteins at high dilutions (0.2 to 2 PM), we employed a fluorometric assay with fluorescamine (FLURAM, Roche Diagnos- tics) utilizing siliconized glassware (29); recoveries were 90 to 100%. For these assays, human apo-A-II was dissolved in the same buffer as the standard. Molar concentrations of apo-A-II were calculated from a molecular weight of 17.4 x 19’.

Lipids Analyses-The phosphorus content, determined by the method of Bartlett (30), was converted into phospholipids by multi- plication by the factor 25. The individual phospholipids were sepa- rated and identified as described previously (23).

Unesterified cholesterol, cholesteryl esters, and triglycerides were analyzed by radioisotopic techniques (23), for which [7-“HIcholesterol (8.7 Ci/mmol), cholesteryl[l-14C]oleate (2.5 mCi/mmol), and glycerol tri[l-‘“Cloleate (55 mCi/mmol)(Amersham/Searle) were used. The radioactivity was measured with a refrigerated Searle Mark III scin- tillation counter. Free cholesterol and cholesteryl esters in HDL were separated and quantitated as already reported (23). Triglycerides in HDL were extracted with 3:2 (v/v) ethanol/ethyl ether with an internal standard of glycerol tri[l-‘“Cloleate and then quantitated by the thin layer chromatography-charring method of Kritchevsky et al. (31), with glycerol trioleate (Applied Science Laboratories) used as a standard. Appropriate corrections were made for the recovery of labeled glycerol trioleate. Specific activities were determined from the known specific activity of the free cholesterol, cholesteryl oleate, and glycerol trioleate and from the concentration of the unlabeled material. For the calculation of the molar concentrations, the molec- ular weights of cholesteryl linoleate (649), unesterified cholesterol (387), and glycerol trioleate (885) were used.

Circular Dichroism-Circular dichroism studies were performed as previously described (32), with a Cary model 6001 spectropolar- imeter provided with a circular dichroism attachment (Cary Instru- ments) and calibrated with an aqueous solution of (d)-lO-camphor- sulfonic acid (Eastman Kodak).

Amino Acid Analyses-Lipid-free proteins were hydrolyzed in 6 N HCl for 24 h at 110°C. Analyses were done in duplicate in a Beckman model 121 amino acid analyzer equipped with an automatic sample injector, scale expander, and integrator. Cystine was quanti- tated as cysteic acid after performic acid oxidation (33).

Other Studies

Ouchterlony double diffusion experiments and agarose gel electro- phoresis were performed as reported earlier (23). Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate was carried out essen- tially according to the method of Weber and Osborn (22). Analytical polyacrylamide gel electrophoresis in 8 M urea (19) was a modification of the procedure described by Davies (21). For transmission electron microscopy of negatively stained specimens, the method of Ohtsuki et al. (34) was used.

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Interaction of APO-A-II with HDL Lipids

Chemicals

The chemicals were reagent grade. Organic solvents were freshly distilled before use. Reagent grade urea (Mallinckrodt, Inc.) was recrystallized from 95% ethanol and deionized through a mixed bed ion exchange resin (Bio-Rad). Fresh urea solutions were always utilized at 10°C to eliminate, or at least to minimize, cyanate forma- tion, and protein carbamylation (35-37). That artifacts were probably not generated during chromatography of the apoprotein in urea was indicated by the following: (a) apo-A-II had electrophoretic mobility corresponding to that of the mother product; (b) no spurious bands were observed; (c) the NH, terminus was not blocked; and (d) no homocitrullin was present in the 6 N HCl hydrolysates of any of the fractions obtained.

TABLE I

RESULTS

In pilot studies, we were able to establish that mixtures of HDL lipids and apo-A-II in a weight ratio of 1, when fraction- ated by CsCl density gradient ultracentrifugation, ultracen- trifugal flotation, and gel permeation chromatography, yielded a good separation of the unreacted constituents from the lipid. protein complexes. In accordance with our studies on apo-A- I (23), we will consider a lipid. protein complex as the product containing both lipid and protein, either banding in isopycnic

gradients or eluting on agarose columns in a position inter- mediate between that of the unreacted protein and lipid. We will refer to the reassembled HDL particles as R-HDL (apo- A-II).

Initial Lipid/Protein Weight Ratio of 1

Initial APO-A-II Concentration of I1 PM-Typical profiles of essentially lipid-free apo-A-II, free lipid, and the lipid. apo-A-II complexes obtained by CsCl density gradient ultra-

60

00 40

40 20

0 0 Jo Bottom TOP

FIG. 1. Profiles of apo-A-II, lipid, and lipid.apo-A-II complexes obtained by density gradient ultracentrifugation. The gradient ex- tends from density 1.45 approximately to density 1.03 g/ml (from left to right). APO-A-II or HDL lipids, or both, were co-sonicated in a total volume of 6.2 ml and centrifuged at a density of 1.006 g/ml at 11°C for 24 h at 39,000 rpm in a Spinco 40.3 rotor, followed by CsCl isopycnic density gradient ultracentrifugation as described under “Materials and Methods.” The number above each band is the average density in grams/ml. A, sonicated apo-A-II (1.2 mg) without lipids (000, typical gradient profile after centrifugation); B, soni- cated HDL lipids (1.2 mg) without apo-A-II; C, apo-A-II (1.2 mg) co- sonicated with HDL lipids at an initial lipid/protein weight ratio of 1.

Comparison ofproperties of R-HDL (apo-A-II) particles with those of native HDL3

APO-A-II was co-sonicated at an initial concentration of 11 PM at a lipid/protein weight ratio of 1 and ultracentrifuged at a density of 1.063 g/ml, as described under “Materials and Methods.” The density of 1.063 g/ml undernatants were then fractionated as outlined under “Materials and Methods.”

Physical parameters Hydrated density, g/ml 1.13-1.08 1.14 S~,,.Z,, Svedberg units N.D.” 2.00b Size, 8, (electron microscopy) 90-200’ 94 k 6d [elm8 “rn x lo-4 -1.89’ -2.20’

[elm, , x m4 -1.87’ -2.15’ Apparent molecular weight, x lo5 0.35-1.29’ 1.75 Electrophoretic mobility (agarose, a1 a1

1%)

Weight per cent

Chemical composition Protein 58 56 Total phospholipids 26.9 26.2

Phosphatidylcholine 22.7g 21.8h*’ Sphingomyelin 2.8 2.3’ Phosphatidylethanolamine Lysophosphaditylcholine 1

1.6 2.1’

Free cholesterol 2.9 2.8 Cholesteryl esters 9.4 12.0 Triglycerides 2.8 3.0

” Highly heterogeneous. b Ref. 25. ’ R-HDL at density 1.13 g/ml contained 64% (90 to 110 A) and 36%

(125 to 150 A); R-HDL at density 1.08 g/ml contained 43% (100 to 110 A), 49% (125 to 150 A) and 7% (200 A). Fifty particles were counted.

” Ref. 34. ’ Degrees. cm*/dmol. ‘A small amount of an R-HDL particle also eluted near the void

volume (see Fig. 2). p May also represent small amounts of phosphatidylserine and

phosphatidylinositol. h Includes 2.4% phosphatidylinositol and 0.6% phosphatidylserine. ’ Ref. 38.

centrifugation are shown in Fig. 1. In the absence of lipids, approximately 90% apo-A-II banded toward the bottom of the tube (Fig. IA); the remainder of the apoprotein was found at density less than 1.15 g/ml due to some residual phospholipid bound to the apoprotein (about 0.5% by weight). The band which floated to the top (Fig. 1C) was identical in position to that of lipids alone (Fig. 1B) and contained no protein. The R-HDL complexes were heterogeneous; the most prominent complex had a hydrated density of 1.13 g/ml at its peak maximum; another component peaked at density 1.08 g/ml (Fig. 1C); a small amount of lipid-free apo-A-II was found toward the bottom of the gradient (see also Table II). This heterogeneity was further documented by electron microscopy of the negatively stained particles (see Table I and Fig. 6A). Varying the pH between 6.6 and 8.6 and the ionic strength of the medium between 10 and 50 mM influenced neither the elution profile obtained on a CsCl density gradient nor the yield and composition of R-HDL. In all cases, results obtained were identical to those reported in Fig. 1C and Tables II and III for the initial apo-A-II concentration of 11 ,uM. Only at low ionic strength (2 mu) were some changes in the elution profiie obtained; the main complex being more homogeneous having a hydrated peak density of 1.18 g/ml; moreover, R-HDL contained approximately 25 and 50% less phospholipid and

free cholesterol, respectively. Agarose column chromatography of the sonicated lipid/

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Interaction of APO-A-II with HDL Lipids

apo-A-II mixtures also gave a good separation of the reacted from the unreacted components. The lipids and the lipid-free apo-A-II (when present) eluted in the void and inclusion volumes of the column, respectively, whereas R-HDL eluted between the unreacted components. The broad elution profile again indicated heterogeneity (Fig. 2).

The properties of the R-HDL (apo-A-II) complexes are shown in Table I. The mean residue ellipticities at both 208 and 222 nm, electrophoretic mobility on agarose, and chemical composition were similar in the reassembled complexes and human serum HDL3. The heterogeneity of R-HDL (apo-A-II) was particularly striking in hydrated density, size, and appar- ent particle weight. An accurate flotation coefficient at density 1.21 g/ml of R-HDL could not be determined because of its heterogeneity.

Initial APO-A-II Concentration Other Than 11 PM---we

next studied the reconstitution of apo-A-II at initial apo-A-II concentrations differing from 11 pM, although retaining an initial lipid/protein ratio of 1 (Tables II and III, and Fig. 3). As shown in Table II and Fig. 3A, at low concentrations (0.2 PM), the amount of apoprotein incorporated into R-HDL was only 28% of the initial protein. As the concentration of the reactants was increased, there was a parallel rise in the amount of apo-A-II recovered in R-HDL up to an initial apo-A-II concentration of 11 PM (Table II and Fig. 3, A to C). Above this concentration, the amount of apo-A-II recovered in R- HDL decreased dramatically, with the majority of the apo- protein now in either lipid-free form or floating at density 1.063 g/ml (see Table II and Fig. 3, C and D). In addition, as the initial concentration of apo-A-II was increased, the het- erogeneity of R-HDL became more pronounced (Fig. 3). With 0.2 pM apo-A-II (Fig. 3A), an apparently homogeneous particle was obtained which had a peak hydrated density of 1.14 g/ml. At an initial concentration of 2 pM apo-A-II, R-HDL exhibited a broad distribution, with banding of hydrated densities at 1.13 and 1.08 g/ml (Fig. 3B). The results were similar to those obtained at 11 pM apo-A-II (Figs. 1C and 3C). At an initial apo-A-II concentration of 108 PM (Fig. 30), there was an R- HDL with a peak hydrated density of 1.20 g/ml which was rich in phospholipids and had essentially no or extremely small amounts of free cholesterol and cholesteryl esters. The other R-HDLs were poorly resolved and peaked between

60 lb0 180 VOLUME, ml

FIG. 2. Elution profile on agarose chromatography of apo-A-II and HDL lipids co-sonicated at an initial lipid/protein weight ratio of 1. APO-A-II and HDL lipids were co-sonicated and centrifuged at a density of 1.063 g/ml at 1l’C for 24 h at 39,000 rpm in a Spinco 40.3 rotor, followed by dialysis of the bottom 2.3 ml against 0.15 M NaCl containing 0.01% EDTA, pH 7.0, and 8% agarose column chromatog- raphy of the undernatant at 8°C (23). VO, column void volume; V,, total column volume (void plus internal volume).

TABLE II

Distribution of apo-A-II in the various fractions obtained after ultracentrifugation of apo-A-ZZ/HDL lipid mixtures at a weight ratio of 1, but at different initial concentrations of each reactant

The solutions of apo-A-II at various concentrations were co-soni- cated with HDL lipids at an initial lipid/protein weight ratio of 1 in a total volume of 6.2 ml (for 0.2 to 11 pM) and 3 ml (for 28 and 108 PM), as described under “Materials and Methods.” For the initial apo- A-II concentrations 0.2 to 2 pM, siliconized glassware was utilized. The sonicated mixtures were centrifuged at a density of 1.063 g/ml as reported in the legend of Fig. 2. The top 1 ml was removed and analyzed for floating apo-A-II; the undernatant was then dialyzed for 1 day against 0.02 M EDTA, pH 8.6, at 8”C, followed by CsCl density gradient ultracentrifugation (see “Materials and Methods”). For the initial apo-A-II concentration of 108 pM, only one-third of the under- natant was applied to the gradient. Lipid-free apo-A-II and the R- HDL complexes are the fractions shown in Fig. 1.

Initial concentration of lipid-free apo-A-II re-

Distribution of apo-A-II

acted with HDL lipids Lipid-free d > at a weight ratio of 1 1.21 g/ml R-HDL Floating at d =

1.063 g/ml

PM

0.2 0.6 2.0 4.0

11.0 28.5

108.0

72 31 27 16 7

50 59

% weight

28 69 73 84 88 35 23

TABLE III

Effect of apo-A-ZZ concentration on the composition of R-HDL (apo-A-II) isolated by CsCl density gradient ultracentrifugation

The solutions of apo-A-II at various concentrations were co-soni- cated with HDL lipids at an initial lipid/protein weight ratio of 1 and centrifuged as described in Table II.

Initial concen- Protein and lipid composition of R-HDL (ape-A-II) tration of apo-

A-II reacted with HDL lip- APO-A-II Phospho- Free cho- Cholesteryl

ids lipids lesterol esters

w !-VT

0.2 6.4 N.D.” 0.2 0.7 0.6 44.3 N.D.” 1.5 5.2 2.0 158.0 N.D.” 7.5 19.9 4.0 360.0 N.D.” 14.0 43.0

11.0 1044.0 491 48.7 141.0 28.5 530.0 384 42.7 111.4

108.0 520.0b 642’ 55.0h 140.0b

n Phospholipids too low to be determined. ’ Analysis does not include R-HDL, which banded at density 1.20

g/ml.

densities 1.10 and 1.063 g/ml. The lipid distribution of the heterogeneous R-HDL particles was unaffected up to an initial concentration of 11 pM apo-A-II. Above this concentration, there was an increased incorporation of cholesteryl esters, free cholesterol, and phospholipids into the lipid. apo-A-II com- plexes (Table III).

Because of the effect that apo-A-II concentration had on lipid binding and because we were aware of the concentration- dependent self-association by this apoprotein (5, 6, 39), we further studied the solution behavior of the lipid-free apo-A- II by circular dichroism. Apoprotein concentration affected the circular dichroic behavior of apo-A-II, as shown by changes in the 222 and 208 nm ellipticities (Fig. 4, top panel). Below 1 PM apo-A-II, a marked decrease in the mean residue ellipticities at both 208 and 222 nm occurred (Fig. 4, bottom panel). At apo-A-II concentrations between 1 and 54 pM, there was a gradual increase in these values; above 54 pM protein, the ellipticity values reached a plateau. This concentration dependence of apo-A-II is in agreement with the circular

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0 100 40 ?-

E %

20 bZ

ZP 0

iE_ WZ

50 20 IO :A

“? v

0 0

200 80

100 40

0 0 Bottom b Ik TOP

FRACTION NUMBER

FIG. 3. Density gradient ultracentrifugation profiles of lipid/pro- tein mixtures sonicated at different initial apo-A-II concentrations. APO-A-II and HDL lipids were co-sonicated and centrifuged at a density of 1.063 g/ml as described in the legend of Fig. 2, followed by dialysis of the undernatant against 0.02 M EDTA, pH 8.6, and CsCl density gradient ultracentrifugation (see “Materials and Methods”). The phospholipid concentrations in A and B were too low to be determined. For initial apo-A-II concentrations of 0.2 and 2 PM, siliconized glassware was used. The initial apo-A-II concentrations were: A, 22 pg or 0.2 PM; B, 216 pg or 2 pM; C, 1.2 mg or 11 pM; and D, 5.7 mg or 108 PM. For the initial apo-A-II concentration of 108 pM,

only one-third of the undernatant was applied to the gradient.

dichroic studies previously published (5, 27), although these reports were carried out in less detail.

Effect of Initial Lipid/Protein Weight Ratios below and above 1

In the following experiments, the initial concentration of apo-A-II was 11 pM.

Initial Lipid/Protein Weight Ratios below I-When apo- A-II was co-sonicated with HDL lipids at an initial lipid/ protein weight ratio less than 1, the hydrated density and lipid composition of the lipid-protein complexes obtained by density gradient ultracentrifugation differed from those at a weight ratio of 1 (Fig. 5; compare with Figs. 1C and Fig. 3C). At an initial lipid/protein weight ratio of 0.2, the complex formed had a peak density of 1.21 g/ml (Fig. 5A). At an initial lipid/protein weight ratio of 0.5 (Fig. 5B), there was a decrease in the amount of R-HDL (peak density, 1.21 g/ml) in favor of heterogeneous complexes banding at lighter densities. Under these conditions, due to overlap, it was not possible to differ- entiate the apo-A-II that was lipid-free from that incorporated into R-HDL; the protein-free lipids were at the top of the tube. Compared to native human HDLZ, all of the lipid. apo- A-II complexes which formed at initial lipid/protein ratios

below 1 had a small complement of lipid (see Table VI). Initial Lipid/Protein Weight Ratios above l-Under these

conditions, the R-HDL (apo-A-II) complexes could be sepa- rated from the lighter particles only by ultracentrifugal flota- tion at density 1.063 g/ml, followed by fractionation of the undernatant by CsCl density gradient ultracentrifugation. At weight ratios above 1, the R-HDLs were only minor compo- nents (Table IV) and exhibited properties that varied consid- erably from those obtained at a lipid/protein weight ratio of 1. For example, at an initial lipid/protein ratio of 4, approxi- mately 70% of R-HDL had a hydrated peak density of 1.070 g/ml, whereas the remainder banded between densities 1.13 and 1.080 g/ml. In addition, R-HDL was markedly heteroge- neous in size, as determined by electron microscopy of the negatively stained particles: 120 to 140 A, 50%; 167 to 200 A, 43%; and 270 A, 7%. The apparent particle weights ranged from 17.4 to 21.8 x 104, with a small amount eluting in the void volume of a gel permeation column containing 8% aga- rose. The R-HDL (apo-A-II) complexes were also rich in phospholipids and free cholesterol compared to those obtained at an initial lipid/protein weight ratio of 1 (see Table VI).

The lighter particles which floated at density 1.063 g/ml were the predominant ones that formed at an initial lipid/ protein weight ratio of 4 (Table IV). These particles exhibited a broad, heterogeneous profile obtained by NaCl isopycnic density gradient ultracentrifugation between densities 1.050 and 1.028 g/ml, with a peak hydrated density of 1.037 g/ml; 14% banded at density 1.024 g/ml and 16% floated at the top of the gradient. Electron microscopy of the negatively stained particles also showed heterogeneity: 150 to 200 A, 17%; 230 to

a, -0.8 -

3 -l.O-

:

Y -l.2-

3 -1.4-

-0.8k 4

I I I ,,I I 20 40 60 ” 90 II0

APO A-II, /LM

FIG. 4. Circular dichroism spectra of lipid-free apo-A-II as a func- tion of concentration. The analyses were performed in 0.02 M sodium phosphate buffer, pH 8.0, at room temperature. Cuvettes of path- length 10 mm were used for 0.3 and 0.7 PM, 5 mm for 1.4 and 2.8 pM, 1 mm for 5.2 to 21.8 pM, and 0.1 mm for 38.6 to 109 PM. The sample spectra at 0.3 and 0.7 pM was 1.5 and three times over background and the dynode voltage did not exceed 0.4. Top panel, spectra at 0.3, 10.9, and 109 pM; bottom panel, plot of mean residue ellipticity 208 and 222 nm versus apo-A-II concentrations.

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Interaction of APO-A-II with HDL Lipids

0 BOitOm

FRACTION NUMBER

1

FIG. 5. CsCl density gradient profiles of apo-A-II and HDL lipids co-sonicated at different initial lipid/protein weight ratios. The ex- perimental conditions are those in the legend of Fig. 1. The initial lipid/protein weight ratios were: A, 0.2; B, 0.5

TABLE IV

Distribution of apo-A-II in the various fractions obtained after ultracentrifugation of apo-A-ZZ/HDL lipid mixtures sonicated at

initial lipid/protein weight ratios of 1 and above, but at a constant concentration of apo-A-II

Solutions of apo-A-II, 11 pM, were co-sonicated with different amounts of HDL lipids and then fractionated by sequential flotation as described in Table II. R-HDL was separated between densities 1.063 and 1.21 g/ml. The lipid-rich particles were fractionated from the density <1.063 g/ml material by ultracentrifugation at density 1.006 g/ml as described under “Materials and Methods.”

Distribution of z$o-A-II Initial lipid/apo- A-II weight ratio d > 1.21 g/ml R-HDL Lipid-:$ parti-

1 2 4

6 0 0

70 weight

94 0 53 47 25 75

300 A, 77%; and 380 to 540 A, 6% (Fig. 6B). Their apparent weights ranged from 8 x lo” to greater than 3 x lo”, as estimated by gel permeation chromatography on a column of Sepharose 4B; 4% of the particles eluted in the void volume. The weight per cent composition, indicative of the predomi- nant lipid nature of these particles, was: protein, 20; phospho- lipid, 62; free cholesterol, 6; cholesteryl esters, 10; and triglyc- erides, 2. Because these particles contained mostly lipid and little protein, they will be referred to as lipid-rich particles.

We next compared the properties of these lipid-rich parti- cles obtained at an initial lipid/protein weight ratio of 4 with those of the HDL lipids alone. Aqueous dispersions of HDL lipids were sonicated and then fractionated by NaCl density gradient isopycnic ultracentrifugation; cholesteryl esters and triglycerides floated at the top of the gradient, whereas the remainder of the HDL lipids banded sharply at a peak density of 1.022 g/ml. The lipid particles isolated at density 1.022 g/ ml contained phospholipids and unesterified cholesterol in a molar ratio of about 4:l (Table V); these proportions have been reported in artificial mixed vesicles (40). By electron microscopy, these fractionated lipid particles appeared as multilamellar vesicles in size ranges significantly larger than the spherical lipid-rich particles (see Fig. 6, B and C). On a Sepharose 4B column, approximately 90% of these vesicles were recovered in the void volume, whereas the remainder eluted near the exclusion volume with apparent particle weights greater than 3 x 10”. In turn, almost all of the lipid- rich particles were eluted in the included volume of the

column indicative of a smaller size in accord with the electron microscopic findings. In addition, the lipid-rich particles had dissimilar hydrated peak densities when compared to the lipid vesicles. In spite of differences in physical properties, lipid vesicles and lipid-rich particles had similar phospholipid and unesterified cholesterol composition (Table V). These findings demonstrate that the interaction with apo-A-II changed sig- nificantly the structural properties of the vesicles obtained from the HDL lipids.

Data Analyses of the Results Obtained for Different Initial Lipid/APO-A-II Ratios

In an attempt to obtain some understanding of the various reassembly data, we examined those obtained at varying initial lipid concentrations while keeping the initial apo-A-II concen- tration constant (Table VI). We recognized the difficulties arising from the analysis of heterogeneous particles. However, we felt that the information, although approximate, could prove useful. We calculated the number of molecules/particle for each component, based on the weight per cent composition of the lipid.apo-A-II complexes and the apparent molecular weight from the peak fraction of the reassembled particles. Based on these calculations, several interesting results emerge from the data summarized in Table VI. The R-HDLs con- tained 4 mol of apo-A-II, while the apparent particle weight increased steadily over the initial lipid/protein weight ratios studied. The number of apo-A-II molecules/particle, there- fore, is not the major determinant of the size of the lipoprotein; this finding is in striking contrast to those described for apo- A-I which, under similar experimental conditions, exhibited a clear limiting effect on HDL size (23). As the apparent particle weight increased, there was enhanced incorporation of choles- teryl esters into the R-HDL particles up to a weight ratio of 1, at which the particles had a chemical composition closer to HDL, (except for a lesser amount of cholesteryl ester). Also, within a weight ratio of 0.5 to 1, R-HDL exhibited a substantial increase in phospholipids from 26 to 46 mol, which correlated well with particle size. In contrast, the small amount of R- HDLs which formed at initial lipid/ape-A-II ratios >l con- tained more phospholipid and free cholesterol and consider- ably less cholesteryl esters and triglycerides compared to native HDL3. The fact that their lipid composition approached that of the HDL lipid vesicles raises the possibility of an artifact in fractionation (compare Table V with Table VI). Up to a ratio of 1, HDL-like particles were predominant; above this ratio, the lipid-rich particles were preferred. The fact that the lipid composition of the lipid-rich particles was similar to

TABLE V

Lipid composition of the HDL lipid vesicles alone and of the lipid- rich particles obtained from an HDL lipid/ape-A-ZZ mixture co-

sonicated at a weight ratio of 4

HDL lipid vesicles were from 4.8 mg of sonicated HDL lipids and were separated from cholesteryl esters and triglycerides by NaCl isopycnic density gradient ultracentrifugation. Co-sonicated mixtures of HDL lipids and apo-A-II (1.2 mg, 11 pM) at an initial lipid/protein weight ratio of 4 were fractionated as described in Table IV; values refer only to the lipid-rich particles obtained from the HDL lipid/ apo-A-II mixture.

Lipid composition

Reactants Separated Phos- FIW

pholipid choles- ‘Fe$fe Triglyc-

tero1 esters erides

% weight

HDL lipids Vesicles 89 9 1.7 0.3 HDL lipids + Lipid-rich 77 8 12.0 3.0

apo-A-II particles

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Interaction of APO-A-II with HDL Lipids 2523

FIG. 6. Electron micrographs of negatively stained (1% sodium obtained at a lipid/protein ratio of 1 as in Table II: B, lipid-rich phosphotungstate) R-HDL (apo-A-II), lipid-rich particles, and HDL particles obtained at a lipid/protein ratio of 4 and fractionated as in lipid vesicles. APO-A-II (1.2 mg, 11 pM) was co-sonicated with HDL Table IV; and C, lipid vesicles isolated from 4.8 mg of sonicated HDL lipids at a lipid/protein weight ratio of 1 or 4 as outlined under lipids as in Table V. “Materials and Methods.” A, R-HDL (apo-A-II) at density 1.13 g/ml

TABLE VI Data analyses of the results obtained at different initial lipid/ape-A-ZZ ratios

The values listed refer to initial concentration of 11 pM apo-A-II analyzed. The apparent molecular weight of R-HDL (apo-A-II) and sonicated with different amounts of lipids. After ultracentrifugation of the lipid-rich particles were obtained from the predominant peak of the lipid/protein mixtures at a density of 1.063 g/ml as described fraction of the particles eluted from agarose columns as described in the legend of Fig. 2, the undernatant was adjusted to a solution under “Materials and Methods.” The weight per cent composition of density of 1.21 g/ml with NaBr and centrifuged for 24 h at 11°C in a protein, phospholipid, cholesterol, cholesteryl esters, and triglycerides 40.3 rotor at 39,000 rpm. The top 1 ml containing R-HDL was is given in parentheses. collected, dialyzed against 0.15 M NaCl with 0.01% EDTA, pH 7, and

Apparent Chemical composition Initial lipid/ape-A-II weight ratio molecular

weight Protein Phospholipid Free cholesterol Cholesteryl esters Triglycerides

x10’ molecules/particle

R-HDL 0.50 1.05 4.2 (69.5) 26.0 (18.6) 5.2 (1.9) 11.2 (6.9) 3.7 (3.1) 0.67 1.12 4.2 (65.2) 31.7 (21.1) 6.4 (2.2) 13.1 (7.6) 3.8 (3.0) 1.00 1.29 4.4 (58) 46.3 (26.9) 9.7 (2.9) 18.7 (9.4) 4.1 (2.8) 2.00 1.58 3.7 (41) 97.9 (46.5) 14.3 (3.5) 18.0 (7.4) 3.3 (1.6) 4.00 2.18 4.3 (34.2) 160.0 (55) 29.3 (5.2) 14.5 (4.3) 3.2 (1.3)

Lipid-rich particle, 4.06 8.40 9.6 (20) 694.4 (62) 130.2 (6) 129.4 (10) 18.9 (2)

Control HDLa 1.75 5.6 (56)” 60.6 (26) 12.7 (2.8) 32.4 (12) 5.9 (3)

o The protein content of HDL, was considered to be due entirely to apo-A-II.

that of the HDL vesicles indicates that the association be- tween the lipids and apo-A-II was merely an adsorptive proc- ess followed by vesicle disruption into heterogeneous lipid- rich particles.

DISCUSSION

In previous studies on the reassembly of apo-A-I, the results supported the conclusion that this apoprotein has an essential role in the structure of human HDL (23). This conclusion was based mainly on the following observations: (a) the in vitro complexation of apo-A-I with lipids was under kinetic control in that the process did not reach thermodynamic equilibrium; (b) apo-A-I generated a lipid. protein complex with properties similar to those of the parent lipoprotein; (c) the number of apo-A-I molecules incorporated into the reassembled HDLs determined their size and structure. If we compare those results with those derived from our current work, it is evident that the mode of complexation of apo-A-II with HDL lipids differs in many respects from that exhibited by apo-A-I. As it

was the case with apo-A-I (23), apo-A-II can probably generate HDL particles through a kinetically driven process under narrowly defined experimental conditions (initial lipid/apo-A- II ratio of approximately 1). However, this apoprotein cannot define particle size for, although the R-HDL particles had the same number of apo-A-II molecules (total, 4), they all carried a different complement of lipid with parallel differences in apparent molecular weight and size (Table VI). In the case of apo-A-I, a clear bimodal distribution in size and molecular weight was observed between the R-HDLs that had incorpo- rated either 2 or 3 apo-A-I molecules (23). Moreover, the complexation of apo-A-I with HDL lipids was reduced signif- icantly at high initial protein concentrations and was favored by dilution. These observations were attributed to the state of self-association of this apoprotein (23). On the other hand, the complexation of apo-A-II with HDL lipid was impaired at both high (108 IJM) and very low (0.2 /.tM) initial apoprotein concentrations. The low recoveries of R-HDL at high initial apo-A-II concentrations may be explained by its state of

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Interaction of APO-A-II with HDL Lipids

oligomerization, which had previously been documented by ultracentrifugal studies (5, 6, 39). In turn, apo-A-II at high dilutions, although monomeric (39), exhibited changes in con- formation, as shown by circular dichroic measurements. Therefore, these results provide further support of the concept that the solution properties of an apoprotein must be known before a meaningful study of its ligand binding can be under- taken (4-6, 39, 41).

REFERENCES

That apo-A-II cannot define the size of the HDL particle is also indicated by the fact that particles resulting from the interaction of apo-A-II and HDL lipids exhibited a much broader distribution than that observed in the reassembly studies with apo-A-I (23). In the current work, when using high initial lipid/ape-A-II ratios, we noted that the lipid-rich particles were the most predominant lipid. protein complexes formed. These particles were characterized by marked heter- ogeneity in size and density and exhibited a lipid composition which closely resembled that of the HDL lipid vesicles pre- pared in the absence of apo-A-II, although differing from the latter in physical properties. These findings suggest that, at high initial lipid/protein ratios, complexation was governed by vesicle geometry which was altered following the interac- tion with apo-A-II.

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Overall, our results indicate that apo-A-II exhibits impor- tant differences from apo-A-I in its mode of interaction with HDL lipids. It is recognized that these two apoproteins differ in chemical properties (2) and in their behavior in solution (42) and at the air-water interface.” Thus, one can assume that these apoproteins will also have a different behavior at the lipoprotein surface. APO-A-II, contrary to apo-A-I, appears incapable of defining the size of HDL, suggesting a difference in structural role between these two apoproteins. We know, for example, that the binding of apo-A-I for lipids is enhanced by the presence of apo-A-II (7,43). It has also been suggested that apo-A-II has a greater affinity for sphingomyelin than does apo-A-I (12). This suggestion, however, is not supported by our studies since the particles assembled with apo-A-II had the same phosphatidylcholine/sphingomyelin ratio as the HDL that was reassembled only in the presence of apo-A-I. Recently, hydridization experiments involving canine HDL and lipid-free human apo-A-II have shown that apo-A-II has a greater affinity for the HDL surface than apo-A-I (44); this observation may be related to the notion that apo-A-I, but not apo-A-II, is readily released from HDL. All of these findings suggest that apo-A-I and apo-A-II have complemen- tary roles in HDL structure. Based on our results, an essential structural role can be invoked only for apo-A-I; this conclusion is supported by the observation that the HDLs of animal species, such as the cow (45), pig (46), or dog (47), contain apo-A-I as their major constituent. At this time, the actual role of apo-A-II in HDL structure remains undefined, except that its presence may add stability to the HDL particle (48). Studies on the molecular behavior of apo-A-II at the hydro- philic-hydrophobic interface, either in model systems or in HDL, are likely to provide useful information on this subject. Studies in this direction are in progress in this laboratory.

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Achnowleclgments-We wish to thank Mr. J. Foreman for amino acid analyses and Drs. M. Ohtsuki and T. Kirchhausen for the electron microscopic studies. We acknowledge the editorial comments provided by Mrs. E. Lanzl, and the typing of this manuscript by Mrs. Karen Kosakowski and Mrs. Rose Scott.

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M C Ritter and A M ScanuAn approach by reassembly techniques.

Apolipoprotein A-II and structure of human serum high density lipoproteins.

1979, 254:2517-2525.J. Biol. Chem. 

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