Conformation of apolipoprotein E both in free and in lipid-bound form may determine the avidity of triglyceride-rich lipoproteins to the LDL receptor: structural and kinetic study

Conformation of apolipoprotein E both in free and in lipid-bound formmay determine the avidity of triglyceride-rich lipoproteins to the LDL

receptor: structural and kinetic study

Alexander D. Dergunov a;*, Elizaveta A. Smirnova a, Aksam Merched b,Sophie Visvikis b, Gerard Siest b, Vladimir V. Yakushkin c;1, Vladimir Tsibulsky c;1

a National Research Center for Preventive Medicine, 10 Petroverigsky street, 101953 Moscow, Russiab Centre du Medicament et Centre de Medecine Preventive, Universite Henri Poincare et INSERM Unite 525, 30 rue Lionnois,

54000 Nancy, Francec Institute of Experimental Cardiology, Russian Cardiology Complex, 3rd Cherepkovskaya street, 15a, 121552 Moscow, Russia

Received 11 October 1999; received in revised form 30 November 1999; accepted 16 December 1999

Abstract

Slow refolding of human apolipoprotein E (apoE) in solution after guanidine- or cholate-induced denaturation followedby dialysis under controlled conditions was investigated using various spectroscopic properties of fluorescein- and dansyl-labeled apolipoprotein molecules. The results suggest that the last phase(s) of apoE refolding in solution include a slow(several hours at 24³C) interconversion of a self-associated òpen' conformer into a more dense `closed' conformer. Thehydrophobic interactions are primarily responsible for the formation of this more compact apoE structure. To visualize thecontribution of apolipoprotein conformation and/or the number of àctive' lipid-bound apoE molecules in the reaction ofbinding to the low density lipoprotein receptor (LDLr) by solid-phase binding assay, the complexes of human plasmaapolipoprotein or recombinant (rec) apoE3 with dipalmitoylphosphatidylcholine (DPPC) or palmitoyloleoylphosphatidyl-choline (POPC) varying in size were used. For seven complexes with plasma protein (four DPPC and three POPCcomplexes), the final phosphatidylcholine (PC)/protein mole ratio ranged from 117 to 279; affinity constant Ka averaged forboth PCs and plotted against this ratio abruptly increased from 3.8U107 to 3.8U108 M31 with a transition midpoint of 150^180 PC/apoE, mole ratio. Two DPPC complexes with rec protein bind much more efficiently. Complexes with both plasmaand rec apoE were able to compete with very low density lipoproteins (VLDL) or low density lipoproteins (LDL) isolatedfrom patients with E3/3 phenotype, for binding to the LDLr. Again, the competition efficiency abruptly increased at theincrease in PC content with a transition midpoint of 130 PC/apoE, mole ratio. The transitions observed both in direct andcompetitive binding assay probably correspond to the abrupt increase in the number of àctive' apoE molecules on thecomplex surface accompanying the change in the size and/or in the shape of the complexes. The efficiency of apoE and apoB

1388-1981 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 1 3 8 8 - 1 9 8 1 ( 9 9 ) 0 0 1 9 6 - 1

Abbreviations: apoB, apolipoprotein B; apoE, apolipoprotein E; apoE/D, dansyl-labeled apoE; apoE/F, £uorescein-labeled apoE;Bu¡er A, 0.02 M sodium phosphate, pH 7.4, 0.15 M NaCl, 0.05% NaN3, 0.01% Na2EDTA, 0.1 mM PMSF; Bu¡er B, 0.02 M Tris-HCl,pH 7.5, 0.15 M NaCl, 0.1 mM EDTA; Bu¡er C, 0.02 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.01% Na2EDTA, 0.05% NaN3 ; Chol,cholesterol ; CM, chylomicrons; DPPC, dipalmitoylphosphatidylcholine; DTT, dithiothreitol ; Gdn-HCl, guanidine hydrochloride; LDL,low density lipoproteins; LRP, LDL receptor-related protein; LDLr, LDL receptor; PMSF, phenylmethylsulfonyl£uoride; POPC,palmitoyloleoylphosphatidylcholine; recapoE, recombinant apoE; TG, triglyceride; VLDL, very low density lipoproteins

* Corresponding author. Fax: +7 (095) 9285063; E-mail : [email protected] Provided technical assistance.

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as the corresponding major ligands in the binding reaction of VLDL and LDL to the LDL receptor was compared. VLDLbind to LDLr following a simple encounter complex model, while LDL binding was characterized by a more complex two-step model with an additional isomerization step. The analysis of the binding data led us to suggest the existence of thecontinuum from several (2^3) apoE molecules on the surface of TG-rich particles that resulted in the increased bindingaffinity, on average 3.5-fold higher, compared to LDL. The existence of a complex equilibrium between aqueous anddifferent lipid-bound forms of apoE is proposed, in particular, the formation of a transient disc-lipoprotein particle structureduring the interaction with LDLr in vivo as well as in LPL-stimulated lipolysis of the lipid phase of the particle. ß 2000Elsevier Science B.V. All rights reserved.

Keywords: Apolipoprotein B; Apolipoprotein E; Very low density lipoprotein; Low density lipoprotein; Low density lipoprotein re-ceptor; Protein structure and refolding; Self-association; Hydrophobic interaction

1. Introduction

Apolipoprotein (apo) E plays a key role in thebinding of triglyceride-rich lipoproteins ^ chylomicraand very low density lipoproteins (VLDL) ^ to lowdensity lipoprotein (LDL) receptor and LDL recep-tor-related proteins (LRP) [1,2]. The conformationand/or concentration of apolipoprotein E on thelipoprotein surface determines the ability of the lipo-protein particle to interact with the receptor: largeVLDL from normolipidemic subjects do not interactwith the LDL receptor while large VLDL from hy-pertriglyceridemics do [3]. We proposed the existenceof cluster organization of amphipathic apolipopro-teins as a result of their secondary self-associationwithin the lipid phase [4]. However, the exact apoEorganization on the surface of the triglyceride(TG)-rich particle has not been elucidated yet, andthe investigation of the apoE structure in solu-tion may contribute towards the solution of thisproblem.

Apolipoprotein E self-association in solution hasbeen described previously [5^8] and we proposed anequilibrium scheme for various apoE structures insolution [5] : oligomer (in aged preparations)3tetra-mer3native or partially denatured monomer3fullydenatured monomer. The structure of apoE in solu-tion might control the dynamics of apolipoprotein-lipid interaction and further re£ect the conformation-al transition(s) during apoE dissociation from thesurface of TG-rich particles during lipolytic degrada-tion [9]. It is not clear whether these conformationaltransition(s) are relatively fast process(es), i.e. canapolipoprotein in dissociated state exist in non-equi-librium state in measurable amounts. The existence

of intermediate non-equilibrium structures could in-duce a `reaction-like' structure of apoE. On the otherhand, apolipoprotein-phospholipid recombinants be-have as stable structures [10], and apolipoproteinconformational change(s) from a free to a lipid-bound structure should be fast process(es). Thepresent study is concerned with: (1) the investigationof refolding kinetics of apoE pre-denatured by gua-nidine hydrochloride or by sodium cholate leading tothe proposal of possible intermediate structure(s)during apoE refolding; (2) the study of the abilityof apoE to interact with the LDL receptor (LDLr)being on the surface of the lipid particles with di¡er-ent geometry, discoidal and spherical; (3) the consid-eration of the contribution of both conformation anddynamics of apoE molecules in a metabolic fate ofdi¡erent apolipoprotein pools as parts of a complexequilibrium between kinetically and structurally dif-ferent structures underlying the metabolism of trigly-ceride-rich lipoprotein particles.

2. Materials and methods

2.1. Subjects and blood sampling

Blood was collected on EDTA from patients at theCenter for Preventive Medicine in Moscow and fromthe healthy laboratory personnel at the Centre deMedecine Preventive in Nancy; all subjects hadapoE 3/3 phenotype.

2.2. Lipoprotein isolation

Very low density lipoproteins (d6 1.006 g/cm3)

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and low density lipoproteins (1.0066 d6 1.063 g/cm3) were isolated from plasma by sequential ultra-centrifugation [11], dialyzed against bu¡er A (0.02 Msodium phosphate, pH 7.4 with 0.15 M NaCl,0.05% NaN3, 0.01% Na2EDTA, 0.1 mM PMSF) at1:100 volume ratio with one change and ¢lteredthrough 0.22 Wm ¢lter (Millipore, USA). Lipopro-teins were stored at 4³C and used within 10 daysafter isolation.

2.3. Apolipoprotein E isolation and labeling

Apolipoprotein E (Mr = 34.2 kDA) was puri¢edfrom pooled human plasma very low density lipopro-teins by gel ¢ltration of delipidated VLDL proteinsin denaturing conditions followed by a¤nity chroma-tography on heparin-Sepharose [12]. The recombi-nant apoE3 (recapoE3, Mr = 40.5 kDA) was ob-tained by single-step a¤nity chromatography onnickel as described [13]. The purity of apolipoproteinwas checked by SDS-PAGE [14] in a 5^20% gel pre-pared in the laboratory. Generally, the purity wasgreater than 95%. The reduction of disul¢de bondsof apoE and blockage of thiol groups by iodoacet-amide (alkylation) were subsequently performed [15].Native or alkylated apoE (5.6 WM) was labeled witheither dansyl chloride (Serva) or with £uorescein iso-thiocyanate (Isomer 1, Sigma) in 0.1 M NaHCO3,pH 9.2 at a dye/protein molar ratio around 10:1.The extent of labeling was varied by varying the in-cubation time which never exceeded 14 h. Unreacteddye was separated by gel ¢ltration on a PD-10 col-umn (Pharmacia/LKB) equilibrated with 0.02 MTris-HCl, pH 7.5, 0.15 M NaCl, 0.1 mM EDTA(bu¡er B) and subsequent dialysis against a 100-fold excess of bu¡er B. Dithiothreitol was added tothe native apoE preparations, at a concentration of1 mM. Samples were ¢ltered through a 0.45 Wm¢lter, HV type (Millipore) and the protein con-centration in the £uorescent-labeled preparationswas determined [16] with native or alkylated apoEas a standard. A molar extinction coe¤cient ofO280 = 43 376 M31 cm31 was calculated based uponthe amino acid content of native apoE. The labelingstoichiometry was calculated using coe¤cients ofO340 = 3400 M31 cm31 for bound dansyl [17] andO495 = 42 500 M31 cm31 for bound £uorescein [18].

2.4. Preparation and characterization ofapoE/dipalmitoylphosphatidylcholine(DPPC) recombinants

Palmitoylphosphatidylcholine and palmitoyloleo-ylphosphatidylcholine (POPC) were purchased fromSigma (St. Louis, MO, USA). Preparation andcharacterization of the complexes were describedin detail in our previous paper by cholate removalprocedure [19]. Bu¡er C (0.02 M Tris-HCl, pH 7.4with 0.15 M NaCl, 0.01% Na2EDTA, 0.05% NaN3)was used throughout this procedure and the initialprotein (plasma apoE or recapoE):PC weight ratioswere within the 1:3 to 1:5 range. Brie£y, after chol-ate was removed from the lipid/apoE/detergent mix-ture by incubation with Bio-Beads, the complexeswere reisolated by gel ¢ltration on a Sephacryl S-300 SF (1.6U62 cm) column with measurements ofoptical density at 280 nm. Protein and lipid recov-eries were generally 70%. The e¤ciency of cholateremoval was greater than 99.5% as estimated froma control experiment by inclusion of a tracer quan-tity of [3H]cholic acid into the initial mixture, i.e.,complexes after Bio-Beads procedure contained lessthan 2 mol% cholate relative to DPPC content. Thechemical composition of the isolated complexes wasassayed as follows: DPPC and POPC were measuredwith a commercial kit (Boehringer Mannheim, Ger-many) and the apolipoprotein content was deter-mined from the absorbance value at 280 nm. Nativegradient PAGE (4^20%) with subsequent immunode-tection of apoE was used to check the dimensionsand homogeneity degree of the complexes. Proteinstandards with the following Stokes diameters [20]were included in each gel: bovine serum albumin(7.1 nm), lactate dehydrogenase (8.2 nm), catalase(9.2 nm), horse ferritin (12.2 nm), and thyroglobulin(17.0 nm), supplied in the Pharmacia Fine Chemicalscalibration kit.

2.5. ApoE denaturation and refolding

Samples for kinetic measurements of apoE renatu-ration were prepared as follows. A mixture of alkyl-dansyl-labeled apoE (apoE/D) and alkyl-£uorescein-labeled apoE (apoE/F) a at 1:1 donor:acceptor mo-lar ratio (donor-acceptor, DF-sample) was denatured

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by incubation in bu¡er B with 4 M guanidine hydro-chloride (Gdn-HCl) or with 60 mM sodium cholatefor 1^3 h at 24³C. For native apoE, dithiothreitol(DTT) at a concentration of 10 mM was added tothe mixture. Samples containing either native or al-kylated apoE/D (donor, D-sample), native or alkyl-ated apoE/F (acceptor, F-sample) or native or alkyl-ated unlabeled apoE (blank) were denatured inparallel. The total apolipoprotein concentration wasidentical in the four samples and equal to 0.6^0.8WM. After guanidine- or cholate-induced denatura-tion, the samples were dialyzed at 4³C for 48 hagainst a 500-fold excess of bu¡er B with 0.1 mMphenylmethylsulfonyl£uoride (PMSF) in dialysisbags (Serva) with a cuto¡ of 12^14 kDa with twochanges of bu¡er. 1 mM DTT was added to thedialysis bu¡er in the case of non-reduced samples.Cholate elimination was enhanced by addition of abile acid adsorbent XAD-2 (Serva) at a concentra-tion 0.2 g/l to the dialysis bu¡er. After dialysis, anadditional incubation step either at 0³C or at 24³Cwas performed. The £uorescent parameters weremeasured at 24³C after varying incubation times us-ing new sample aliquots taken freshly at each timepoint to avoid photobleaching.

2.6. LDL receptor binding assay

The protocol described in detail elsewhere [21,22]was adopted to evaluate the binding e¤ciency ofapoE-containing complexes, isolated lipoproteinsand whole sera. Two models were applied to describethe binding of VLDL and LDL to the LDL receptor.

(1) A simple model 1 of `the encounter complex'formation, where the overall reaction e¤ciency iscontrolled by the frequency of collisions betweenthe receptor and ligand molecules:

L�R 3k11

k21

LR �1�

(2) A more complex two-step model 2 with aninitial fast formation of lipoprotein-receptor complexfollowed by a slow conformational transition of thecomplex:

L�R 3k12

k22

LR 3k32

k42

LR� �2�

In Eqs. 1 and 2 L, R, LR and LR* denote lipo-

protein, receptor, the initial and ¢nal lipoprotein-re-ceptor conformers, respectively. k11 and k12 are theassociation rate constants of the ¢rst step in the en-counter complex and in the slow isomerization mod-els, respectively, k21 and k22 are the correspondingdissociation rate constants, k32 and k42 are the asso-ciation and dissociation rate constants of the secondstep in the slow isomerization model. The ¢rst sub-script denotes the direction of the partial reactionwhile the second one belongs to the model number.Ka11 ( = k11/k21) and Ka12 ( = k12/k22) are the a¤nityconstants for the encounter complex formation andfor the ¢rst step in the slow isomerization model,Ka22 ( = k32/k42) and Ka(tot) ( = Ka12(1+Ka22)) arethe a¤nity constants for the second step and overallreaction in the slow isomerization model, Ka is thea¤nity constant obtained under quasi-equilibriumconditions. The primary kinetic data were ¢tted toEq. 3 which assumes a slow irreversible step at thebinding to the surface-localized receptor molecules[23]:

B � a�13e3t=d 1� � bUt �3�where B is a receptor-bound lipoprotein measured asapoB in terms of optical density units, a = k11ULaURaUd1, b = aUkon, d1 = (k11ULa+ k21)31 andLa and Ra are the total lipoprotein and receptor con-centrations, kon is the rate constant for the irreversi-ble step. It has been shown in a separate experimentthat in our conditions there was a large excess of theligand relative to the receptor. The binding kineticswas studied at several (5^6 points) concentrationsof VLDL and LDL and k11 and k21 values werecalculated from the linear plots d31

1 vs. La. In theslow isomerization model the k42 value was calcu-lated as an intercept from the initial linear part ofthese plots followed by the secondary treatment ofthe data according to Eq. 4 to calculate Ka12 and k32

values [24] :

1d31

1 3k42� 1

k32� 1

Ka12Uk32U

1La

�4�

The equation is valid under the assumptions of: (1)equilibrium conditions, i.e. k22Ek32 relation shouldexist ; and (2) a large excess of the lipoprotein overthe receptor.

The direct binding data obtained at quasi-equili-brium, i.e., after incubation for 2 h at 37³C, were

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¢tted to a model with one class of high a¤nity bind-ing sites (Eq. 5) or, additionally, taking into consid-eration the non-saturable binding (Eq. 6):

B � BmaxU10P�X

1� 10P�X �5�

B � BmaxU10P�X

1� 10P�X � KnsU10X �6�

Both for complexes and lipoproteins, B and Bmax

denote the binding at the speci¢c ligand concentra-tion and the maximum number of binding sites, re-spectively, in terms of optical density units at 492nm; P = log Ka with Ka as an association constantat quasi-equilibrium, X = log Co, Co is the total li-gand concentration in terms of apoB or apoE con-tent; Kns in Eq. 6 describes the contribution of non-saturable binding. Generally, Eq. 6 was used whenthe Kns value exceeded (0.5UBmaxUP).

From competition study with apoE-containingcomplexes with pre-determined Ka values, the a¤nityconstants Kc

a for the binding of lipoproteins and awhole plasma to the LDLr at quasi-equilibrium werecalculated from

Kca �

Ka�complex�UIC5031�B� �7�

IC50 is the concentration of the competitor that re-sults in 50% inhibition of the lipoprotein or a wholeplasma binding and this parameter was determinedas an x scale intercept of the linear ¢t of the data inthe coordinates ln�B=B0�=�13B=B0� vs. log[Etotal],where B and B0 are the concentrations of boundlipoproteins measured as apoB in the presence andin the absence of the complex at the concentrationEtotal.

2.7. Fluorescence study

Fluorescent measurements on apoE refolding werecarried out at 24³C either on Shimadzu RF-540 orAminco-500 spectro£uorimeters equipped with athermostated cell holder with 0.1³ accuracy. For en-

ergy transfer measurements, the excitation slit wasset at 2 nm together with the emission slit at 4 nm(Aminco-500) or both set at 5 nm (Shimadzu RF-540). The spectrum of the blank sample was sub-tracted from those of the £uorescent-labeled samplesand the data were stored in an interfaced computer.The energy transfer e¤ciency E, determined from thesensitized £uorescence of the acceptor is given by

where I and A are £uorescence intensity and absorb-ance values at the excitation and emission wave-lengths indicated as subscript and superscript forthe samples indicated in parentheses. In this calcula-tion procedure, the donor-acceptor sample is excitedat the donor absorption band (345 nm) and the£uorescence intensity is measured at the emissionwavelength of the acceptor (530 nm). The £uores-cence intensity values of the donor and acceptor,excited directly at 345 nm, are subtracted from theformer value as proposed by Borochov-Neori et al.[25].

The £uorescence anisotropy values r of the apoE/F£uorescence were corrected on the polarization in theemission beam with the aid of the G-factor [26]. Thescattered light was further subtracted from the sig-nal. Excitation and emission wavelengths were 490and 520 nm respectively.

The quenching of apoE/F £uorescence by I3

anions was performed by sequential addition of 6 Wlaliquots from 4 M KI stock solution (containinga few crystals of Na2S2O3 to prevent I3 oxidation)to 400 Wl of the sample in 0.3U1.0 cm cuvette. Thequenching parameters, i.e. the fraction of £uorescentresidues accessible to the quencher f and the Sern-Volmer quenching constant KSÿV, were determinedaccording to Eq. 9 [4] :

I0

I03I� 1

f �Q�KSÿV� 1

f�9�

where I0 and I are the £uorescence intensities in theabsence and in the presence of a quencher at a givenconcentration Q.

E�%� � 100��I530345�DF�3I485

345�DF��I530345�D�=I485

345�D��3I530490�DF��I530

345�F�=I530490�F��=I530

490�DF��A345�D�=A490�F� �8�

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2.8. Analytical methods

Protein concentrations in VLDL and LDL weredetermined by the method of Lowry et al. [27]. Cho-lesterol and triglyceride contents in plasma and iso-lated lipoproteins were determined using standardenzymatic methods. ApoE and apoB in plasma andlipoproteins were quanti¢ed by turbidimetry andnephelometry using a Cobas autoanalyzer and aBehring nephelometer, respectively. ApoE phenotypewas determined by isoelectric focusing of delipidatedplasma samples in vertical mini-gel followed by im-munoblotting with a polyclonal anti-apoE rabbitantiserum [28]. The validity of this procedure wascon¢rmed by restriction isotyping [29] and the datacoincided for 27 out of 28 samples passed throughboth procedures.

2.9. Data treatment

Conventional methods were used for the calcula-tion of means, and standard error of means (S.E.M.).Di¡erences between mean values were evaluated witha two-tailed Student's t-test.

3. Results

3.1. ApoE refolding in solution

The self-association of apoE to form supramolec-ular structures was studied after guanidine- or aftercholate-induced apolipoprotein denaturation fol-lowed by its refolding through dialysis and subse-quent incubation at either 0³C or 24³C. Structuralchange(s) during apolipoprotein incubation were fol-lowed by measuring the e¤ciency of non-radiativeenergy transfer E between dansyl- and £uorescein-labeled apoE molecules, the intensity I and anisotro-py r of the £uorescence of the apoE/F and apoE/Dand the quenching parameters of the apoE/F £uores-cence by I3 anions. The temperature dependence ofconformational parameters could be indicative of theinvolvement of hydrophobic interactions into proteinfolding [30,31]. Results obtained with both alkylatedand reduced apoE were combined for analysis. Thelabeling stoichiometry in three separate labeling pro-cedures varied between 4.1 and 12 moles of dye/moleprotein with dansyl and between 3.6 and 4.9 moles ofdye/mole protein for £uorescein.

Table 1ApoE refolding at two di¡erent temperatures

Incubation (h) Incubation temperature

0³C 24³C

Energy transfer E (%) 0 30.0 þ 5.1 (4)3 23.8 þ 6.5 24.5 þ 6.2 (4)6 33.6 þ 12.7 (4) 34.3 þ 8.3 (3)

Fluorescence anisotropy r 0 0.146 þ 0.0025 (5)3 0.146 þ 0.0025 (5) 0.133 þ 0.004 (4)6 0.140 þ 0.004 (4) 0.135 þ 0.003 (4)

Fluorescence intensity I (%) 0 100 (5)3 95.3 þ 5.3 (4) 102.8 þ 7.4 (5)6 84.2 þ 4.7 (4) 95.7 þ 9.1 (4)

Accessibility degree f 0 0.79 þ 0.12 (3)6 0.71 þ 0.05 (3) 0.45 þ 0.07 (3)

Quenching constant KSÿV (M31) 0 11.0 þ 2.9 (3)6 12.6 þ 2.9 (3) 12.9 þ 3.0 (3)

ApoE/F (320 nM), apoE/D (320 nM), their mixture at 1:1 molar ratio and unlabeled protein (in all cases the total protein concentra-tion was equal to 640 nM) were denatured by incubation in bu¡er B with 60 mM sodium cholate for 3 h at 24³C. DTT at a concen-tration of 10 mM was added for non-alkylated samples. Samples were dialyzed at 4³C for 48 h against XAD-2-containing bu¡er Bwith 0.1 mM PMSF in 500-fold excess with two changes (1 mM DTT was added for non-alkylated samples). After dialysis, the sam-ples divided into two pools were continued to incubate at 0³C and 24³C. Energy transfer e¤ciency, apoE/F £uorescence intensity andanisotropy values as well as parameters of apoE/F £uorescence quenching by I3 anions were measured at di¡erent times at 24³C. Re-sults are given as mean þ S.E.M. The number of separate experiments is indicated in parentheses.

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For cholate-induced denaturation, the e¤ciency ofenergy transfer was around 29% during 6 h incuba-tion both at 0³C and 24³C (Table 1). The £uores-cence anisotropy did not change during the incuba-tion at 0³C whereas the mean r value decreasedsigni¢cantly from 0.146 to 0.135 (Table 1). Similarresults were obtained for refolding after denaturationby Gdn-HCl (data not shown). The high energytransfer and anisotropy values measured after dialy-sis suggest that apoE self-association takes place insolution in agreement with previous studies [5^8].

After incubation for 6 h at 24³C the decrease ofthe £uorescence intensity of apoE/F (average 4%)was less pronounced than after incubation at 0³C(average 16%, Table 1). The £uorescence intensityof apoE/D decreased by an average 13^17% after6 h incubation, although the in£uence of the temper-ature on this parameter was not statistically signi¢-cant (data not shown). Similar changes of £uores-cence intensity were obtained for apoE refoldingafter denaturation by Gdn-HCl (data not shown).The time- and temperature-dependent changes ofthe £uorescence intensity and anisotropy suggestthe occurrence of slow conformational change(s) ofapoE during the last phase(s) of refolding, when theapolipoprotein is in a self-associated state. This hy-pothesis was tested by studying the apoE/F £uores-cence quenching by I3 anions. The values of Stern-Volmer quenching constant KSÿV and of the degreeof chromophore accessibility f are given in Table 1.

At the two incubation temperatures studied, a heter-ogeneous mode of quenching was observed. The de-gree of accessibility of the £uorescein groups linkedto apoE molecule (an average value of 0.79 immedi-ately after dialysis) did not change after incubationat 0³C for 6 h whereas it signi¢cantly decreased to0.45 after incubation at 24³C. The incubation step atthese two temperatures had no in£uence on thequenching constants of the chromophores still acces-sible to I3 anions (Table 1). Similar observationswere made for apoE refolding monitored by KSÿV

and f parameters after Gdn-HCl-induced denatura-tion (data not shown). Therefore, the slow conforma-tional changes occurring during apoE refolding seemto decrease the exposure of the £uorescein groups tothe aqueous phase; the self-associated structure ofapolipoprotein changed from an òpen molten glob-ule' to a more condensed state with the formation ofa hydrophobic `core' shielded from the water phaseduring the last phase of refolding.

3.2. Binding of apoE-containing complexes to theLDL receptor

The complexes of human plasma apoE with DPPCand POPC (two initial PC/protein weight ratios wereused in the case of DPPC complexes) as well as thecomplexes of recE3 with DPPC were prepared andcharacterized by composition and by size (Table 2).The elution pro¢les were heterogeneous and, after

Table 2Relationship between the binding e¤ciency of apoE-containing complexes to the LDL receptor and complex composition

Initial PC/proteinweight ratio

Final PC/proteinweight ratio

Complexdiameter (nm)

Complexyield (%)

Binding sites(Bmax, A492)

A¤nity constant(Ka, 108 M31)

E/DPPC1 3.3:1 2.87:1 11.3 52�1�2� 0.52 0.6E/DPPC2 3.3:1 3.34:1 14.4^15.1 0.69 1.2E/DPPC3 5:1 4.52:1 14.4^15.1 71�3�4� 0.82 2.8E/DPPC4 5:1 6.01:1 s. ; 15.1 0.78 3.9E/POPC1 3.3:1 2.61:1 11.3 66�1�2�3� 0.92 0.4E/POPC2 3.3:1 3.25:1 s. ; 15.1 0.94 0.8E/POPC3 3.3:1 3.41:1 s. ; 14.7^15.8 1.46 1.7recE3/DPPC1 3.3:1 4.33:1 14.1 27�1�2� 1.21 þ 0.14 (4) 15.6 þ 0.5 (4)*recE3/DPPC2 3.3:1 4.49:1 s. ; 15.1^15.8; 14.1 1.06 þ 0.12 (4) 25.2 þ 4.1 (4)

The complex yield was calculated for a whole peak in the elution pro¢le obtained by gel ¢ltration and the complex size was measuredby native gradient gel electrophoresis. Only one value is given for a single peak whereas the multiple values belong to the complexsub-species comparable in their intensities. s. denotes the presence of the large sub-species remaining on the start position.The mean values from two independent measurements are given. Binding parameters for recombinant apoE3 are mean þ S.E.M., n = 4.*Signi¢cantly di¡erent (P6 0.06) in column vs. recE3/DPPC2.

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deconvolution by eye, two or three sub-types of thecomplexes marked 1^4 in increasing size order werecollected for the subsequent work. The yield of thecomplexes calculated for a whole peak (1+2 or1+2+3) varied and it seemed to increase in the pre-dictable manner: recE3/DPPC6 plasma apoE/DPPC(low PC/protein ratio)6 plasma apoE/POPC (thesame low ratio)6plasma apoE/DPPC (high PC/pro-tein ratio). The primary reason for this seems to bethe di¡erent e¤ciency of apolipoprotein solubiliza-tion by the phospholipid which in turn is controlledby the aggregation state of the apolipoprotein. Thesmallest complexes with the ¢nal PC/protein weightratio 2.6^2.9 were highly homogeneous in size irre-spective of phosphatidylcholine used with the meandiameter as 11.3 nm as estimated by native gradientgel electrophoresis that coincides well with the di-mension of these small discoidal complexes measuredby a variety of methods [19]. The dimension andinhomogeneity degree of the complexes with DPPCincreased up to 15.1 nm at increasing PC/proteinweight ratio to 4.5 and the largest subspecies of thecomplex did not enter into the gel at a further in-crease of this ratio up to 6. The POPC-containingcomplexes behave in a similar manner as the ratioincreased up to 3.4 (Table 2). For seven complexeswith plasma protein the ¢nal PC/protein mole ratiovaried between 117 and 279. The complexes of re-combinant apoE3 with DPPC seemed to be indistin-guishable in size from the analogous complexes withplasma protein.

The e¤ciency of the binding of DPPC- and POPC-containing complexes to the LDL receptor increasedat increasing the size of the complexes (Table 2) andthe a¤nity constant Ka, averaged for both phos-phatidylcholines, changed abruptly 10-fold from3.8U107 to 3.8U108 M31 with a transition midpointvalue of 164 mole PC/mole apoE (Fig. 1) that mightre£ect the change in the lipid package [19] and/orapolipoprotein conformation [32] as it had been ob-served for apoA-I-containing complexes at increasingthe complex size. For both phosphatidylcholines,larger complexes with plasma apoE seemed to inter-act with the larger number of the binding sites thatexcludes the possibility of steric hindrance and con-trasts with the lattice model of the binding of muchlarger VLDL and LDL particles in the cell-surfaceassay proposed earlier [23,33]. Compared to plasma

protein, lipid-bound recombinant apoE bound to theLDL receptor much more e¤ciently with the Ka val-ues (1.60 þ 0.04)U109 (n = 4) and (2.50 þ 0.40)U109

M31 (n = 4) at DPPC/protein molar ratios 239 and248, respectively (Table 2). Complexes with bothplasma and recombinant apoE were able to competewith isolated VLDL for the binding to the LDL re-ceptor. Again, the competition e¤ciency expressed as1/IC50, with IC50 as the concentration of the complexthat resulted in 50% inhibition of VLDL binding,

Fig. 1. E¡ect of particle size and/or composition on the particlea¤nity to the LDL receptor and on their ability to competewith VLDL in the binding reaction. ApoE-containing com-plexes varying in size were prepared by the cholate removalprocedure and subsequent gel ¢ltration. In the direct bindingassay, the serially diluted complexes were added to the LDLr-coated microtiter wells, incubation was performed at 37³C for2 h and apoE still bound to the well surface after several washeswas measured with the aid of antiE-polyclonal antibody. In thecompetitive assay, the initial binding of very low density lipo-proteins isolated from a patient with hypertriglyceridemia (plas-ma TG as 307 mg/dl) to immobilized LDLr was performed at37³C for 2 h at a ¢xed concentration corresponding to the mid-dle range of the titration curve. After discarding the unboundmaterial and well rinsing, the serially diluted complexes wereadded and an additional 2 h incubation and subsequent wash-ings were performed. The residual VLDL content was measuredas apoB with the aid of antiB-peroxidase-conjugated polyclonalantibody. The primary binding data at quasi-equilibrium weretreated as described in Section 2. The a¤nity constant valuesKa as the reciprocal values of apoE molar concentration aregiven for complexes with DPPC or POPC as open or closedcircles, respectively; the values of IC50 as the apoE concentra-tion in the complexes that resulted in 50% inhibition of VLDLbinding are given for complexes with DPPC or POPC as openor closed triangles, respectively. The curves are the sigmoidal¢ts for Ka and IC50 dependences with X values correspondingto Y50 as 164 þ 38 and 135 þ 6, PC/apoE molar ratios, respec-tively.

BBAMCB 55576 10-2-00


abruptly increased at increasing the phosphatidylcho-line content with a transition midpoint of 135 molePC/mole apoE (Fig. 1). The transitions observedboth in the direct and the competitive binding assayprobably correspond to the abrupt increase in thenumber of àctive' apoE molecules on the surfaceof the complexes accompanying the change in thesize and/or in the shape of the complexes.

3.3. VLDL and LDL binding to the LDL receptor

The ability of three di¡erent VLDL and LDLpreparations isolated from hypertriglyceridemic pa-tients with E3/3 phenotype to interact with theLDL receptor was tested by the solid-phase bindingassay. Both kinetic and quasi-equilibrium (2 h at37³C) measurements of VLDL and LDL bindingwere performed to visualize the possible contributionof steric hindrance(s) into the interaction of ligandswith surface-immobilized receptors [34]. The bindingcurves at VLDL and LDL content within the 0.3^9and 3^90 Wg apoB/ml range, respectively, did notreach plateau values at incubation times up to 20 h(data not shown) probably due to slow parking/re-parking of ligand between the closely spaced receptormolecules with the rate constant kon [23]. This e¡ectwas taken into consideration at the treatment of theprimary data according to Eq. 3; the kon values didnot di¡er signi¢cantly for VLDL and LDL bind-ing and were equal to (2.27 þ 0.39)U1036 and(1.95 þ 0.42)U1036 s31, respectively. The results ofthe treatment of a single representative set ofVLDL and LDL binding are presented in Fig. 2and summary data using two binding models aregiven in Table 3. For both types of lipoproteins,the values of the association and dissociation rateconstants were 2^3 orders of magnitude lower com-pared to the corresponding constants for the reactionin solution and coincided well with k11 and k21 valuesas 8.8U103 M31 s31 and 2.5U1034 s31 for antigen-antibody interaction for hemispherical antigen-

Fig. 2. The binding of VLDL and LDL to the LDL receptor ina solid phase assay. VLDL and LDL were isolated from pa-tients with plasma TG as 255 and 579 mg/dl, respectively. Theprimary kinetic data on speci¢c binding measured within a 20 hinterval were ¢tted to Eq. 3; the partial rate constants were cal-culated from the linear plots d31 vs. total ligand concentrationas predicted by the complex encounter model in the case ofVLDL binding (a) and from the secondary linear plots accord-ing to Eq. 4 as the best ¢t of the data of LDL binding withinthe slow isomerization model (b). For both dependences R val-ues were higher than 0.95 and P6 0.005.

Table 3Kinetic and equilibrium study of VLDL and LDL binding to the LDL receptor

Encounter complex model

k11 (104 M31 s31) k21 (1034 s31) Ka11 (108 M31) Ka (108 M31)

VLDL 1.19 þ 0.48* 2.15 þ 0.12* 0.58 þ 0.24*;# 1.88 þ 0.55*LDL 0.08 þ 0.03 2.75 þ 0.07 0.03 þ 0.01$ 0.54 þ 0.17

Slow isomerization model

k32 (1034 s31) k42 (1034 s31) Ka12 (108 M31) Ka22 (108 M31) Ka(tot) (108 M31)

LDL 2.46 þ 0.57 2.38 þ 0.22 0.21 þ 0.09 1.03 þ 0.20 0.42 þ 0.17

Lipoproteins isolated from plasma of hypertriglyceridemic patients ([TG] = 384 þ 99 mg/dl, n = 3) were tested in the solid-phase bindingassay. The data were treated as indicated in the legends to Figs. 1 and 2 and the results are given as means þ S.E.M.*P6 0.05 for VLDL vs. LDL comparison in column by one-tail t-test in the encounter complex model.#P6 0.05 for VLDL vs. LDL comparison in column by one-tail t-test in the slow isomerization model.$P6 0.07 for Ka11 vs. Ka12 comparison at LDL binding by one-tail t-test.

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coated wells [35] due to di¡usion reaction control.For VLDL binding, the a¤nity constant valuesKa11 derived from the concentration dependence of1/d1 (Fig. 2a) within model 1 with binding as therate-limiting step, did not di¡er within the experi-mental error from the Ka values measured at quasi-equilibrium. The characteristic reaction timed* = (k11U[VLDL]+k21) derived from kinetic param-eters and plasma VLDL content as 0.1 mg apoB/mlwas equal to W390 s = 6.5 min. For LDL bindingwithin this model, however, the Ka value exceededthe Ka11 value 18 times at a mean level, which ledus to consider a more complex two-step model 2which included, besides the fast ligand-receptor com-plex LR formation with the a¤nity constant Ka12, amore slow conformational transition into LR* con-former with the equilibrium constant Ka22 [23]. Theprimary LDL binding data ¢tted well to this model(Fig. 2b) and the above-mentioned di¡erence be-tween kinetic and equilibrium values disappeared(Table 3). The Ka values obtained correspondedwell to published data [36]. The decrease of the k11

value at (k21/k32) times for LDL binding within mod-el 1 may be suggested to occur due to the equilibriumcondition k32Ik21 [24]. The isomerization step (1)was characterized by the characteristic timed* = (k32+k42)31W2070 sW35 min that exceeds con-siderably the d* value for VLDL binding, i.e. thisstep was indeed rate-limiting and 2 h incubationwas a good approximation to equilibrium conditionsboth for VLDL and LDL binding; (2) did not result

in the additional stabilization of LR* conformer asKa22W1. So, both kinetic and equilibrium measure-ments revealed a 2.8^3.5 increase in Ka(VLDL) rela-tive to Ka(LDL).

The direct binding data were compared to the re-sults from the competition study using the displace-ment of pre-bound isolated lipoproteins or a wholeserum by the complexes of apoE with saturated(DPPC) or unsaturated (POPC) phosphatidylcholine.The primary data on the in£uence of the E/DPPC4

complex on the binding of two separate VLDL andLDL samples in logit/log coordinates are given inFig. 3 and the data are summarized in Table 4.Both for VLDL and LDL particles the Kc

a valuesderived from the competition study with theE/DPPC4 complex exceeded 4^5 times at a meanlevel the values obtained with the E/POPC2 complexthat coincided well with the ratio of the a¤nity con-stants for these complexes (Table 2). A great di¡er-ence was obtained for the a¤nity constant valuesderived from the competition and direct bindingstudy: at a mean level, the Kc

a values exceeded theKa values for the binding of VLDL, LDL and wholesera 8, 15 and 3 times with the E/POPC2 complex,while for the E/DPPC4 complex the correspondingdi¡erences were 34, 72 and 8 times.

4. Discussion

4.1. `Molten globule' state of apoE in self-associatedform(s)

In this paper we studied the refolding and self-association of apolipoprotein E towards its native

Fig. 3. Competition between VLDL or LDL and apoE/DPPC4

complex for binding to the LDL receptor. The data for VLDL(a,b) and LDL (O,R) samples isolated from two patients aregiven for comparison.

Table 4Lipoprotein and plasma binding to the LDL receptor fromcompetition and direct binding study

Kca (108 M31) Ka (108 M31)

with E/POPC2 with E/DPPC4

VLDL 22.6 þ 0.2 (2) 95 þ 28 (2) 2.8 þ 0.0 (2)*;$

LDL 16.4 þ 0.4 (5) 79 þ 19 (5)* 1.1 þ 0.3 (5)*;$

Plasma 6.6 þ 0.7 (4)#;3 19.0 þ 0.2 (4)*;#;3 2.3 þ 0.3 (5)*;$;3

Kca and Ka are a¤nity constant values derived from competition

and direct binding study.Signi¢cantly di¡erent: (1) in row vs. E/POPC2 (*) or E/DPPC4

($); (2) in column vs. VLDL (#) or LDL (3).

BBAMCB 55576 10-2-00


structure after guanidine- or cholate-induced denatu-ration. We carried out measurements of the e¤ciencyof energy transfer in a mixture of dansyl- and £uo-rescein-labeled apoE molecules and of the intensity,anisotropy and quenching of the apoE/F £uorescenceby I3 anions. The donor-acceptor pair selected isoften used for the measurement of distances betweenmolecules and/or their geometry [17,18,37]. We meas-ured the sensitized acceptor £uorescence instead ofthe donor £uorescence quenching as the latter pa-rameter is more in£uenced by various additional fac-tors [38]. The e¡ective energy transfer process de-tected at apoE concentrations lower than 0.6 WM(Table 1) is indicative of apolipoprotein self-associa-tion, as di¡usion-controlled transfer does not occurat such low acceptor concentration, but only above1033 M [38]. Moreover, the use of apoE at a highdegree of labeling (4^12 moles of dye/mole protein)enabled the measurement of variations of the distan-ces between the centers of èmitting spheres', i.e. self-association, rather than the variations in the geome-try of the protein molecule at low labeling [37]. Itshould be noted that the r values obtained either athigh (Table 1) or low degree of protein labeling by£uorescein (6 1 mole of dye/mole protein) [5] did notdi¡er from each other, i.e. £uorescence depolariza-tion due to the close proximity of several £uoresceingroups was negligible.

We postulate that the larger decrease of the £uo-rescence intensity measured at 0³C compared to 24³Cduring apoE/F refolding after guanidine- or cholate-induced denaturation is suggestive of the existence ofintermediate refolding state(s) of the self-associatedapoE structure. Incubation at 0³C favors an òpen'structure for the apolipoprotein E tetramer while at24³C a transition of this conformer to a `closed' stateoccurred and an additional step as the transition be-tween two tetramer forms can be introduced into theequilibrium scheme for the apoE structure in solu-tion. Among the force(s) involved in the stabilizationof the apoE supramolecular structure in solution,hydrophobic interactions are likely to be involvedas suggested by the temperature e¡ects on the time-dependent changes of the apoE/F £uorescence inten-sity (Table 1). Hydrophobic interactions between ali-phatic radicals are more stable at room temperaturecompared to 0³C, due to the endothermic transitionassociated with the radical transfer from water to anon-polar solvent [30]. The temperature dependenceof hydrophobic interactions also accounts for thedenaturation of proteins at low temperatures [31].Ion-pairing might further contribute to apoE self-association, as apolipoprotein E cross-linking withwater-soluble carbodiimide (`zero-length' cross-link-er) induced the formation of a tetramer [39]. Thepredominant role assigned to hydrophobic interac-

Scheme 1. Equilibrium scheme describing the transitions between the various self-associated forms of apoE in solution and the modeof interaction of lipid-bound forms of apolipoprotein with the LDL receptor. The apoE forms in aqueous phase are depicted in bold,the lipid-bound forms in italics. LPL, lipoprotein lipase; LDLr, LDL receptor. The dimensions of the two-end arrows denote the rela-tive a¤nities of the partial binding reactions.

BBAMCB 55576 10-2-00


tions is further supported by the changes of the £uo-rescence anisotropy and of the accessibility of £uo-rescein groups to a water-soluble quencher whichwere more pronounced at 24³C than at 0³C. Theanisotropy value decrease cannot be attributed tointensity changes observed, as quantum yield de-crease associated with a concomitant decrease ofthe excited state lifetime would increase the anisotro-py value. As the e¤ciency of energy transfer was thesame at both temperatures, the formation of a hy-drophobic `core' in the apoE self-associated structuredoes not modify the distance between centers ofèmitting spheres', i.e. of individual apoE moleculesin the tetramer. No appreciable self-association ofapoE into higher oligomer structures occurs duringthe 6 h incubation period. The length of time neces-sary to reach equilibrium for various £uorescent pa-rameters has not been established yet. The r valuereached its equilibrium value after 3 h incubation at24³C, while the £uorescence intensity of apoE/F de-creased during the time interval studied.

Thus, for the ¢rst time for apolipoprotein E, theexistence of a partially disordered, self-associatedapoE tetramer as a structure dominating at the laststep(s) of apoE refolding in solution is suggested.This structure seems to possess the structural proper-ties common with the `molten globule' state such asthe presence of a pronounced secondary structure,high compactness without a rigid packing inside amolecule, and increased £uctuations of protein sidechains as well as of larger parts of a molecule, com-pared to the native state [40]. The `molten globule'state seems to be a rather general structure for otherexchangeable apolipoproteins such as apoA-I [41,42],apoA-I Milano [42] and apoA-II [43]. It can bespeculated further that apoE, when exchanged be-tween di¡erent lipoprotein particles through thewater phase, exists in a partially disordered, self-as-sociated state closely resembling the `molten globule'structure observed by us; a similar suggestion hasbeen made for apoA-I binding to the lipid phase[41]. How could both òpen' and `closed' tetramerforms of apoE be related to the two-domain natureof this apolipoprotein described by us and others[5,7,8]? The C-terminal domain is responsible for lip-id binding whereas the receptor-binding region is lo-cated in the N-terminal domain that belongs to the4-helix bundle family of structures [44] and its mono-

mer structure is stabilized by intra- and interhelicalsalt bridges. Both domains seem to be related struc-turally to each other [45]. We speculate that fast self-association of apoE in solution mediated by hydro-phobic interactions between individual C-domains isfollowed by a much slower rearrangement of theòpen' tetramer into the `closed' tetramer, the hydro-phobic interactions again being involved. However,the slow formation of the `right' local structures dueto the formation of salt bridges between monomerchains could also contribute to apoE folding. On theother hand, the N-domain of apolipoprotein in alipid-bound form may òpen up' into the most ener-getically favored structure on the lipoprotein surface.

4.2. Conformation and dynamics of apoE are the keydeterminants in the interaction of lipoproteinswith the LDL receptor

The supramolecular structure of apoE in solutionmight be determinant for the e¤ciency and mode ofapolipoprotein binding to the surface of lipoproteinsand the extent of apoE secondary self-association ina lipid phase might in turn control the interaction ofapoE-containing particles with the LDL receptor orLRP. The apoE interaction with the LDL receptor isnot understood as a cooperative process, rather wesuggest the major contribution of the increased num-ber of the àctive' apoE molecules into the increasedbinding of large complexes; each apolipoproteinmolecule on the surface of an apoE-containing com-plex seems to interact independently with the recep-tor molecule. The contribution of àctive' apoE mol-ecules [46] and surface apolipoprotein concentration[47] into lipoprotein binding to the LDL receptor hasbeen proposed by others.

The similar dependence of the binding e¤ciencyboth for DPPC and POPC complexes on the apoEcontent in the isolated complexes (Fig. 1, Table 2)strongly suggests for both phosphatidylcholines qual-itatively analogous e¡ects of apoE on the PC/proteinstoichiometry, distinct apolipoprotein conformationand the complex dimension as had been observedfor apoA-I-containing complexes [48]: the authorssuggested, ¢rst, that common apolipoprotein A-Icon¢guration de¢nes particle size class and, second,the di¡erent lipids alter the particle size distribution.In our case, for apoE-containing discoidal com-

BBAMCB 55576 10-2-00


plexes, we suggest an increase of the number of apo-lipoprotein molecules and/or a change in apoE con-formation at the increase of complex dimension. Inthis relation it is interesting to note that in one mol-ecule of LDL receptor several repeats can interactwith several copies of apoE [49] which may increasethe binding e¤ciency of large complexes comparedto small ones.

The dimension of the ligand regions inside VLDLand LDL particles able to interact with the LDLrcan be estimated with the following assumptions:(1) the steric hindrances are the predominant factorsthat contribute into the e¡ective values of the asso-ciation and dissociation rate constants at LR com-plex formation [50] and the k21 value is determined,in turn, by the reassociation rate with the immobi-lized receptor molecule [34]; (2) the equation describ-ing the contribution of the rotational di¡usion intothe interaction of a small active center on the macro-molecule with the surface-immobilized receptor maybe applied [51] :

ko � koe1

16Z� rs

rA�3 �10�

where ko and koe are di¡usion forward reactive rateand fundamental rate constant which represents thedi¡usion rate constant for reactants of equal radius,rs is the active center radius, rA the macromoleculeradius. Then

Ka11�VLDL�Ka12�LDL� �

ko�VLDL�ko�LDL� �

rs�VLDL�UrA�LDL�rs�LDL�UrA�VLDL��

3 �11�

and, taking into consideration the a¤nity constantvalues (Table 3) and the ratio of the mean dimensionof VLDL and LDL particles as 1.7, the relationrs(VLDL)/rs(LDL)W2.5 was obtained. This valuecoincides well with the mean apoE content inVLDL particles as 2^3 apolipoprotein moleculesper particle and the increased area of the clusterfrom closely located 2^3 apoE molecules on theVLDL surface compared to a single reaction zonein LDL apoB is assumed to determine the increasedVLDL binding e¤ciency.

ApoE molecules seem: (1) to be the principal li-gand in the interaction of VLDL particles with the

LDL receptor; (2) to contact each other with thecluster formation on the VLDL surface. Our directand competition binding data (Table 4) may addi-tionally support this assumption, as apoE-containingcomplexes, when present in a whole serum or beingadded in a competition assay, may (1) `donate' addi-tional apolipoprotein molecules to the lipoproteinparticle where they may possess a conformation dif-ferent from that in the discoidal particle (the di¡erentconformation of apoE in discoidal and spherical par-ticles has been observed [52]); and/or (2) form thetransient tertiary complex LDLr/apoE-disc/lipopro-tein particle. In the latter case, the model of twoligands, i.e. lipoprotein particle and apoE-containingdisc, independently interacting with the receptor mol-ecule may not be valid.

The irreversible nature of the formation of theapoA-I/DPPC complex suggested by Klausner et al.[53] and the increased stability of this apolipoproteinwithin the discoidal complexes compared to apoA-Iin solution [54] seem to contradict the suggestionabout apoE `donation' from disc to lipoprotein par-ticle; at the same time, the stability of apoE in sol-ution and in discs seemed to be identical while theaccessibility of Gdn-HCl-binding sites for lipid-bound apoE was lower compared to apolipoproteinin solution [55]. The latter, together with the de-creased accessibility of tryptophan residues ofVLDL apolipoproteins (apoB, apoE, apoC) relativeto apoB in LDL [4], may be additional evidence ofthe `cluster' hypothesis of apoE organization on theVLDL surface. The formation of the transient com-plex of the discoidal (nascent) and native (mature)lipoprotein particles may resemble the structures or-iginated from the triglyceride-rich lipoprotein par-ticles during their lipolysis. The principal possibilityof the existence of this transient complex followsfrom the existence of a small fraction of HDL withprebeta mobility [56] acting as the most e¤cient met-abolic compartment at early stages of the complexreverse cholesterol transport pathway. The lowest in-crease of Kc

a relative Ka values for plasma in compe-tition assay (Table 4) may also indicate the pre-exis-tence of these transient complexes both for VLDLand LDL particles in plasma.

The cluster organization of the amphipathic apo-lipoproteins on the VLDL surface has been sug-gested earlier by us [5] as well as the lipolysis regu-

BBAMCB 55576 10-2-00


lation by the free fatty acids as natural detergentsformed during triglyceride degradation [4]. In thisstudy we further develop the idea of the existenceof apoE-rich clusters which determine the e¤ciencyof the interaction of VLDL with LDLr and the equi-librium scheme describing the existence of variousself-associated forms of apoE in solution can be ex-tended now to include the lipid-bound forms of theapolipoprotein (Scheme 1). The main suggestionwhich follows from this study is the possible directrelation between apoE surface density and the abilityof VLDL particles to interact with LDLr, which willbe veri¢ed in the paper that follows. Additionally,experiments on the visualization of protein-proteincontacts on the surface of native and partially li-polyzed VLDL particles are in progress now as wellas experiments to visualize the possible existence oftransient complexes of VLDL with apoE-containingdiscoidal HDL particles.

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