Báo cáo Y học: Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions pot

Eur J Biochem 269, 5939–5949 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03319.x Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions Matthew A Perugini1, Peter Schuck2 and Geoffrey J Howlett1 Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; 2Division of Bioengineering and Physical Science, ORS, OD, National Institutes of Health, Bethesda, MD, USA We describe sensitive new approaches for detecting and quantitating protein–lipid interactions using analytical ultracentrifugation and continuous size-distribution analysis [Schuck (2000) Biophys J 78, 1606–1619] The new methods were developed to investigate the binding of human apolipoprotein E (apoE) isoforms to size-fractionated lipid emulsions, and demonstrate that apoE3 binds preferentially to small lipid emulsions, whereas apoE4 exhibits a preference for large lipid particles Although the apparent binding affinity for large emulsions is similar (Kd % 0.5 lM), the maximum binding capacity for apoE4 is significantly higher than for apoE3 (3.0 and 1.8 amino acids per phospholipid, respectively) This indicates that apoE4 has a smaller binding footprint at saturation We propose that apoE isoforms differentiate between lipid surfaces on the basis of size, and that these differences in lipid binding are due to a greater propensity of apoE4 to adopt a more compact closed conformation Implications for the role of apoE4 in blood lipid transport and disease are discussed Traditional methods for monitoring protein–lipid interactions have employed a variety of lipid systems, including emulsions, phospholipid vesicles and phospholipid micelles [1–6] However, the methodologies and heterogeneity of lipids employed in these studies not allow a thorough investigation into the effect of particle size on protein binding This may be important to examine, given that earlier studies have demonstrated that apolipoprotein E (apoE) isoforms differ in their propensity to bind to small and large lipoproteins [7–10] Experimental approaches that can therefore measure and quantitate binding to sizefractionated lipid particles may provide insight into the role of blood lipid transport proteins, such as apolipoprotein E, in lipid metabolism and disease Human apolipoprotein E is a key component of plasma lipoproteins, exchanging reversibly between chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and a subclass of high density lipoprotein (HDL) [11–13] It serves as a high-affinity ligand for a number of cell surface receptors, thereby mediating the uptake of cholesterol and other complex lipids into the cell [12,13] The three common isoforms of apoE, designated apoE2 (C112/C158), apoE3 (C112/R158) and apoE4 (R112/R158), differ by single amino acid substitutions at positions 112 and 158 [13–15] ApoE2 is linked to type III hyperlipoproteinemia and has low affinity for the low density lipoprotein (LDL) receptor [13,16] ApoE3 is the most common isoform and is associated with normolipidemia [13,17], while the apoE4 isoform is independently associated with an increased risk for atherosclerosis [17,18] and late-onset Alzheimer’s disease [19,20] Recent studies compare the structure–function relationships of the apoE isoforms, including their stability [21], self-association in the presence and absence of phospholipid [4], and their ability to bind preferentially to different lipoprotein classes [7–10] ApoE is composed of two independently folded domains The 10 kDa COOH-terminal domain (residues 225–299) possesses high lipid affinity, while the 22 kDa NH2-terminal domain (residues 1–191) binds weakly to lipid and mediates receptor interactions [13] In the absence of lipid, the NH2terminal domain of apoE3 forms an elongated four helicalbundle, stabilized by hydrophobic contacts and intra- and inter-helical salt bridges [22] The substitution of cysteine for arginine at position 112 in apoE4, results in an additional salt-bridge between Glu109 and Arg112 and the displacement of Arg61 from the surface of the four-helix bundle [8] The displaced Arg61 side chain forms an interdomain saltbridge with Glu255, an interaction that is critical for directing the preference of apoE4 to bind large VLDL particles [7] In contrast, the apoE3 isoform binds preferentially to smaller HDL particles, with residues NH2terminal to position 244 implicated as the primary HDL binding determinants [9,10,23] Recently, a number of studies have shown that the 22 kDa NH2-terminal structure of apoE reorganizes upon interaction with lipid [3,24–28] It has been proposed that the NH2-terminal domain of apoE undergoes conformational changes, converting from a closed receptorinactive conformation in the absence of lipid, to an open Correspondence to M Perugini, Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia, 3010 Fax: + 61 39347 7730, Tel.: + 61 38344 5911, E-mail: perugini@unimelb.edu.au Abbreviations: apoE, apolipoprotein E; Myr2Gro-PCho, dimyristoylglycerophosphocholine; EggPtdCho, egg yolk phosphatidylcholine; F, fraction; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; TO, triolein; VLDL, very low density lipoprotein (Received 25 July 2002, revised October 2002, accepted 17 October 2002) Keywords: Alzheimer’s disease; apolipoprotein E; open conformation; lipid binding; analytical ultracentrifugation Ó FEBS 2002 5940 M A Perugini et al (Eur J Biochem 269) receptor-active conformation in the presence of phospholipid [13,27] Evidence in support of this model is provided by recent lipid binding studies employing synthetic lipid emulsions and intact apoE4, its 22 kDa NH2-terminal fragment, and the 10 kDa COOH-terminal fragment [28] At a low surface concentration of protein, the binding enthalpy of intact apoE4 was consistent with the sum of the enthalpies for the 22 kDa and 10 kDa derivatives, indicating that both NH2- and COOH-terminal domains bind to the emulsion surface [28] At saturation, however, the enthalpy for intact apoE4 was similar to that of the 10 kDa fragment, suggesting that only the COOH-terminal domain of intact apoE4 interacts with the emulsion surface [28] It is not known whether the apoE3 isoform displays a similar phenomenon on the surface of lipid particles In the present study we develop methods for the characterization of lipid emulsions of well-defined composition but different particle size We compare the binding of apoE3 and apoE4 to small and large lipid particles, and show that apoE4 has a greater capacity than apoE3 to bind large lipid emulsions These results suggest that at saturation, apoE4 binds primarily in a more compact conformation, whereas apoE3 adopts an expanded conformation on the lipoprotein surface phosphate, pH 7.4 Phospholipid and triacylglycerol concentrations were determined using enzymatic spectrophotometric phospholipid and glycerol assay kits (Roche) Peptide synthesis and purification ApoE(263–286) peptide (SWFEPLVEDMQRQWAGLV EKVQAA, Mr ¼ 2818) [30–32], was synthesized by automated solid phase methods on an Applied Biosystems 431A synthesizer, starting with HMP resin (NovaBiochem) and using Fmoc amino acids (AusPep) The peptide was cleaved from the solid support with trifluoroacetic acid and deprotected with piperidine Before purification, the crude peptide mixture was washed and precipitated in ice-cold diethyl ether, filtered, then solubilized in 100 mM ammonium bicarbonate, pH 7.8, containing 10% (v/v) acetic acid The peptide was purified by reversed-phase FPLC on a 3-mL Resource column with a 10 mL, 0–100% gradient, of acetonitrile containing 0.1% (v/v) trifluoroacetic acid Fractions containing pure peptide, as assessed by matrixassisted laser desorption ion-spray mass spectrometry (Finnigan Mat) were pooled, lyophilized, solubilized in distilled water and stored at )20 °C Expression and purification of apolipoprotein E3 and E4 EXPERIMENTAL PROCEDURES Materials Dimyristoylglycerophosphocholine (Myr2Gro-PCho) and egg phosphatidylcholine (EggPtdCho) were purchased from Sigma, St Louis, MO, USA and triolein [1,2,3-tri(cis-9octadecanoyl)glycerol; TO] from Nu-Chek Prep, Elysian, MN, USA Human apoE3 and apoE4 were expressed in Escherichia coli as fusion proteins with glutathione S-transferase using the pGEX-3X plasmid, and purified as described previously [4] The purity of both apoE3 and apoE4 as assessed by SDS/ PAGE was estimated to be > 98% The molar masses of purified apoE3 and apoE4 (34 245 Da and 34 297 Da, respectively) determined by electrospray mass spectrometry agree well with theoretical values and known differences between the amino acid compositions of the isoforms [15] Emulsion preparation and fractionation Emulsions were prepared as described previously [29] with some modifications A mixture of : TO/Myr2Gro-PCho or : TO/EggPtdCho in chloroform (1.0 or 10.0 mgỈmL)1 total lipid) was dried down under nitrogen, desiccated overnight, and resuspended in 10 mL of 0.1 M sodium phosphate buffer, pH 7.4 The suspension was sonicated at 34 °C using a probe sonicator (Soniprep 150, MSE Scientific Instruments, Sussex, England) at an amplitude of 10 microns for · (with 30 s intervals) under a constant stream of nitrogen The dispersion was extruded 10–12 times through · 100 nm polycarbonate filters using a LiposoFast hand-held microextruder (Avestin Inc., Ottawa, Ontario, Canada) The density of the extrudate was increased to 1.038 gỈmL)1 by the addition of sucrose (10 gỈdL)1) and the sample layered beneath 0.1 M sodium phosphate buffer, pH 7.4 The sample was centrifuged at 110 000 g for 35 in a Beckman 70.1 Ti rotor and model L8-70 ultracentrifuge After centrifugation, the top 1.5 mL was collected (unfractionated) and the density adjusted to 1.018 gỈmL)1 with solid sucrose (5 gỈdL)1), before adding a 0–5% (w/v) linear sucrose gradient prepared in 0.1 M sodium phosphate, pH 7.4 The sample was then centrifuged at 4500 g for h in a Beckman SW-40 rotor and model L8-70 ultracentrifuge Fractions (1.0 mL) were collected from the bottom of the tube using a peristaltic pump and dialyzed exhaustively against 50 mM sodium Dynamic light scattering Dynamic light scattering experiments were conducted with a Protein Solutions DynaPro instrument with MSTC200 microsampler (Protein Solutions, Charlottesville, VA, USA) Samples, suspended in 50 mM sodium phosphate, pH 7.4, were centrifuged for in a microcentrifuge to remove dust particles, and 20 lL sample was inserted in the cuvette with the temperature control set to 20 °C The light scattering signal was collected at 90°, and diffusion coefficients and Stokes-radii of the emulsion fractions were calculated with the instrument software Flotation velocity Flotation velocity experiments were performed using a Beckman model XL-A analytical ultracentrifuge Prior to centrifugation, apoE and emulsion samples were exhaustively dialyzed (> 20 h) against 50 mM sodium phosphate, pH 7.4 Samples (300–400 lL) and reference (320–420 lL) solutions were loaded into a conventional double-sector quartz cell and mounted in a Beckman An-60 Ti rotor Experiments were conducted at 20 °C and at a rotor speed of 5000 r.p.m Data was collected in continuous mode, at a single wavelength (230 nm or 250 nm), time interval of 360 s and a step-size of 0.003 cm without averaging Multiple scans at different time points were fitted to a single species or Ó FEBS 2002 Binding of apolipoprotein E3 and E4 to emulsions (Eur J Biochem 269) 5941 to a continuous size distribution (see below) using the program SEDFIT (which is available at www.analyticalultra centrifugation.com) Solvent densities were measured at 20 °C in an Anton Paar model DMA 02C precision density meter equipped with a water bath, or computed using the program SEDNTERP [33], kindly supplied by D Hayes (Magdalen College, NH, USA), T Laue (University of New Hampshire, Durham, NH, USA) and J Philo (Alliance Protein Laboratories, Thousand Oaks, CA, USA) Partial specific volumes () of apoE3 isoforms (0.732 mLỈg)1) and v apoE(263–286) (0.738 mLỈg)1) were calculated from amino acid composition [33] Size distribution analysis The size distribution of noninteracting lipid emulsion particles can be calculated as a flotation coefficient distribution, c(sf), according to Eqn (1): Z aðr; tÞ ffi cðsf ÞLðsf ; D; r; tÞdsf ð1Þ where a(r,t) denotes the observed optical density at radius r and time t, c(sf) denotes the differential flotation coefficient distribution, L(sf,D,r,t) denotes the solution to the Lamm equation [34], calculated with an adaptation of the moving frame of reference method [35] to flotation velocity, taking into consideration the rotor acceleration phase One feature of boundary modeling with Eqn (1) is that it allows interconversion of the flotation coefficient distribution to a molar mass distribution via the Stokes-Einstein and Svedberg equation [36], upon consideration of the spherical shape and the size-dependent particle density of the polydisperse solutes For the functional dependence between the density and molar mass of the fractionated emulsion particles, we assumed a spherical monolayer of Myr2Gro-PCho surrounding a core of TO, supported by transmission electron microscopy (data not shown) Consequently, the relationship between density of the particles as a function of particle mass was determined using values  for M and v reported by [29], calculated from the ratio of phospholipid to triacylglycerol assuming each phos˚ pholipid molecule occupies an area of 60 A2 in the monolayer surface, and that each triacylglycerol mole˚ cule occupies a volume of 1610 A3 in the particle core [37,38] Linear regression least squares analysis of these data yields the relationship:  v ẳ 1:042 ỵ 0:0191 ẵM=108 ị1=3 2ị Similarly, linear regression analysis of size-fractionated emulsions comprised of EggPtdCho and TO yields the relationship:  v ẳ 1:014 ỵ 0:0264 ẵM=108 ị1=3 3ị Subsequently, the molar mass distribution of Myr2GroPCho/TO and EggPtdCho/TO emulsion fractions were calculated using Eqns (2) and (3), respectively For the mixtures of emulsion particles and protein,  because of the unknown contribution of the protein to the v, a flotation coefficient distribution was calculated by approximating the diffusion with an average diffusion coefficient measured by dynamic light scattering Because of the size of the emulsion particles, diffusional broadening of the flotation profiles is not very large, and variation of the diffusion coefficient throughout the distribution can be considered a second order effect However, this method does not allow the transformation of the flotation coefficient distribution in a molar mass distribution To prevent an ill-conditioned analysis when performing continuous size-distribution analysis with many species, a regularization technique was employed that selects the most parsimonious distribution of species that fits the data within a predetermined confidence limit Consistent with observations in previous studies [4,36], this resulted in smooth distributions However, in contrast to earlier studies with sedimentation coefficient distributions of proteins, for which maximum entropy regularization seemed advantageous because of its potential to produce sharp peaks for discrete mixtures [4,36,39], we found the Tikhonov-Phillips regularization with second derivative functional more useful, because it avoids possible oscillatory artifacts known to be encountered with the maximum entropy method for broad distributions [40] Furthermore, in order to obtain a high parsimony and stability of the distribution, we applied a high confidence limit of P ¼ 0.95 Unless stated otherwise, all size distributions were solved on a grid of 300 radial values between the meniscus and bottom, a confidence level of P ¼ 0.95, frictional ratio (f/f0) ¼ 1.0 and at a resolution (N) of 100 sedimentation coefficients between )1.0 S and )1000 S, respectively This resulted usually in residuals with rms errors < 0.01 Values of f/f0 > 1.0 led to significantly poorer fits, consistent with the spherical shape of the emulsion particles For Monte-Carlo statistical analysis, 1000 synthetic data sets were generated, based on the best-fit continuous size distribution, each with different normally distributed noise For each point in the distribution, the mean and the quantiles enclosing 95% of the values from the analyses of the simulated distributions were determined Apolipoprotein-emulsion binding assay Samples of emulsion alone, protein alone, and emulsion plus protein at various apoE concentrations were centrifuged using conventional quartz cells in a Beckman model XL-A analytical ultracentrifuge for up to h, at a rotor speed of 5000 r.p.m and a temperature of 20 °C Estimates of the signal due to free protein (relative to the emulsion alone control) were calculated from the optical density in the infranatant averaged over a radial range of 0.1 cm in the plateau region using data from the final radial scan The average optical density due to free protein was converted into concentration units via a fivepoint standard curve The concentration of bound protein was calculated based on the measured free and the known total amount of protein The apparent dissociation constant (Kd) and maximum binding capacity (Bmax) were estimated on the basis of a Langmuir isotherm, by plotting the amount of free protein (Pf), against total phospholipid concentration (PC) multiplied by the ratio of free to bound protein (Pf/Pb) [2,6,41]: Pf ẳ PCPf =Pb ịBmax Kd 4ị Data was processed and fitted using the program SIGMAPLOT 5942 M A Perugini et al (Eur J Biochem 269) Ó FEBS 2002 RESULTS Sucrose gradient fractionation of lipid emulsions Synthetic lipid emulsions comprised of TO and Myr2GroPCho were prepared by sonication, pressure extrusion, and fractionated by sucrose gradient ultracentrifugation [29] To initially examine the size-fractionated lipid emulsions, phospholipid and triacylglycerol concentrations of each fraction (F) were determined by enzymatic spectrophotometric analysis and the major lipid-containing samples, F2 to F6, were characterized by dynamic light scattering (Fig 1) The relationship of the particle radius, calculated from the measured diffusion coefficient obtained by dynamic light scattering, to the TO/Myr2Gro-PCho molar ratio demonstrates successful sucrose gradient fractionation of the lipid emulsions (Fig 1) The larger particles show a higher TO/Myr2Gro-PCho ratio, as predicted for a model emulsion comprised of a phospholipid monolayer surrounding a triacylglycerol core Based on dynamic light scattering data, the fractionation procedure yielded individual fractions of particles with radii in the range of 38–52 nm Flotation velocity analysis of fractionated lipid emulsions The solution properties of the major emulsion fractions were further characterized by analytical ultracentrifugation Figure shows flotation velocity data of fractions 2, and at 360 s intervals A significant time-dependent broadening of the flotation boundary is observed in each, suggesting the emulsion fractions are moderately heterogeneous This assertion is supported by the poor fits (rmsd ¼ 0.0574) and nonrandom distribution of residuals, when for example, the data for fraction is fitted assuming a single species with the average diffusion coefficient as determined independ- Fig Flotation velocity of Myr2Gro-PCho/TO fractionated lipid emulsions Absorbance at 250 nm is plotted as a function of radial position (open circles) for fraction (A), fraction (B) and fraction (C) at t ¼ 78–120 The solid lines represent the continuous sizedistribution best-fits Insets: Residuals (DA) are plotted as a function of radial position (cm) Fig Dynamic light scattering of size-fractionated lipid emulsions The TO/Myr2Gro-PCho molar ratio of emulsion fractions (F) 2, 3, 4, and is plotted vs the particle radius determined by dynamic light scattering The solid line represents the linear regression best-fit, describing the relationship between TO/Myr2Gro-PCho molar ratio (m) and particle radius (r) as, m ¼ 0.18r ) 4.9 ently by dynamic light scattering (data not shown) Similar observations were made with fractions 2, 3, 5, and We therefore sought a method to characterize the residual polydispersity In a previous study [29], dc/dt analysis [42] was employed to determine the apparent flotation distribution function g(s*) of fractionated lipid emulsions In particular for large particles and when using absorbance optical ultracentrifuge data, this method is intrinsically limited due to artificial broadening that is introduced by the finite time-difference between the scans considered for dc/dt analysis [43] Therefore, in the present study we took advantage of the continuous size distribution method c(s) and c(M) for direct boundary modeling [36] Although the direct boundary Ó FEBS 2002 Binding of apolipoprotein E3 and E4 to emulsions (Eur J Biochem 269) 5943 method for the apparent flotation coefficient distribution ls-g*(s) does not generate artificial broadening, the c(s) and c(M) methods have the additional advantage of the deconvolution of diffusion effects Furthermore, compared to more traditional approaches, such as the van HoldeWeischet method [44], we have recently demonstrated that better resolution and more detailed distributions can be obtained by c(sf) analysis for determining the size distributions of fractionated lipid emulsions [45] The c(sf) analysis best fits for fractions 2, and are shown in Fig (solid lines), which result in a random distribution of residuals (Fig 2, insets) and low rmsd values (< 0.005) when compared to fits assuming a single species The resulting c(sf) size-distributions for the major emulsion fractions, including fractions 2, and 6, are shown in Fig The data indicate that the size-fractionated emulsions have wellseparated size-distributions, albeit with some degree of overlap The continuous size distributions are broader with increasing fraction size, suggesting that the larger fractions, F5 and F6, are more polydisperse than the smaller fractions, F2 and F3 This may be an artifact of the fractionation process, as the emulsions are harvested smallest to largest following sucrose gradient ultracentrifugation The c(sf) distribution for unfractionated emulsions is also shown, demonstrating a high degree of heterogeneity, as expected, and a double maxima in c(sf) at approximately 200 S and 550 S (Fig 3) Similar c(sf) distributions were obtained when the emulsions were prepared and fractionated at a 10-fold higher total lipid concentration of 10 mgỈmL)1 (data not shown) Furthermore, c(sf) analysis was also employed to demonstrate that the fractionated lipid emulsions were stable over a period of days, at pH values in the range of 4.0–10.0, at temperatures of 5–35 °C, and in the presence of up to M NaCl (data not shown) Continuous mass, c(M), distributions for the major Myr2Gro-PCho/TO fractions were also determined The molar masses at the ordinate maximum of c(M) for each fraction are presented in Table 1, demonstrating that the emulsions range from 9.4 · 107 Da for fraction 2, to 7.00 · 108 Da for fraction Assuming spherical particles, supported by transmission electron microscopy (data not shown), the calculated particle radii at maximum c(M) correspond to 34 nm to 67 nm for fractions and 6, respectively, in the range of IDL-VLDL particles (Table 1), and the dynamic light scattering results (Fig 1) The values in Table are slightly higher than those obtained by dynamic light scattering (Fig 1), an effect attributed to the skewed continuous size distributions especially for the larger particles (Fig 3) Interaction of ApoE(263–286) with lipid emulsions Fig Continuous size-distribution flotation velocity analysis of sizefractionated lipid emulsions Calculated c(sf) from Eqn (1) is plotted vs flotation coefficient, sf, for fractions (F) 2–6 and unfractionated lipid emulsions The c(sf) distribution of the unfractionated lipid emulsions has been arbitrarily scaled We initially examined the binding of a synthetic peptide comprising residues 263–286 of human apoE to the fractionated lipid emulsions ApoE(263–286) is amphipathic in nature, and has previously been reported to bind to Myr2Gro-PCho bilayers [31] and SDS micelles [32], a common lipid-mimetic Figure shows the continuous flotation, c(sf), distribution of emulsion fraction in the absence and presence of apoE(263–286), calculated with a fixed diffusion coefficient of 0.46 · 10)7 cm2Ỉs)1 Relative to the control, the flotation rate of fraction after the addition of 1.0 lM peptide is significantly reduced, accompanied also by an increase in the area under the distribution curve (Fig 4A) These changes are attributed to peptide binding to the emulsion particles Monte-Carlo analysis demonstrates that the observed increase in area under the curve and shifts to lower values of sf in the presence of peptide are Table Hydrodynamic properties of Myr2Gro-PCho/TO size-fractionated lipid emulsions Lipid emulsions composed of Myr2Gro-PCho and TO were fractionated and characterized by size-distribution analysis as described in the Materials and methods The symbols used are sf, flotation coefficient taken from the ordinate maximum of the best-fit c(sf) distribution calculated according to Eqn (1) (Fig 3); M, molar mass taken from the  ordinate maximum of the best-fit c(M) distribution (data not shown); v , partial specific volume calculated from M according to Eqn (2); Rs, particle  radii, calculated assuming a spherical particle and using the experimentally determined values for M and v (as above) Fraction # TO:Myr2Gro-PCho molar ratio sf (S) M (· 108 Da)  v (mLỈg)1) RS (nm) 2.05 2.62 3.18 3.86 4.29 166 333 506 664 795 0.94 2.3 4.00 5.60 7.00 1.061 1.067 1.072 1.076 1.079 34 46 55 62 67 5944 M A Perugini et al (Eur J Biochem 269) Ó FEBS 2002 Fig Binding of apoE(263–286) peptide to Myr2Gro-PCho/TO emulsion fraction The amount of bound apoE(263–286) (symbols + solid line) is plotted as a function of free protein (lg/mL) The concentrations of Myr2Gro-PCho and TO in lipid emulsion fraction are 110 lM and 390 lM, respectively Binding data was obtained by analytical ultracentrifugation using the direct binding assay as described in Experimental procedures Inset: Linearized plot of the binding data for apoE(263–286) (symbols) shown in panel A The solid line represents the linear least-squares fits to the data according to Eqn (4), where the y-intercept and slope equate to the apparent Kd and Bmax, respectively (Table 2) Fig Continuous size-distribution analysis of fraction in the presence and absence of apoE(263–286) peptide (A) The c(sf) distribution calculated using an invariant D ẳ 0.46 Ã 10)7 cm2ặs)1 is plotted as a function of flotation coefficient for Fraction alone (solid line, no symbols); fraction + 1.0 lM apoE(263–286) (solid line, open symbols) and fraction + 10.0 lM apoE(263–286) (solid line, solid symbols) (B) Results of Monte-Carlo statistical analysis distributions, calculated from 1000 synthetic data sets to a confidence level of P ¼ 0.95 The lower (0.025) and upper (0.975) quantiles are depicted as dashed lines, enclosing the mean distribution (solid lines) for fraction alone (labelled 1), and fraction + 1.0 lM apoE(263–286) peptide (labelled 2) statistically significant, and cannot be attributed to noise affecting the data analysis (Fig 4B) At a 10-fold higher peptide concentration of 10.0 lM, a greater decrease in flotation rate is observed, indicating the peptide binds to the emulsion particles in a saturable manner (Fig 4A) However, although c(sf) analysis shows detailed changes of the size-distribution upon peptide binding, it is not directly possible to quantify the observed binding for the calculation of a binding constant and maximum capacity Nevertheless, this can be accomplished by a direct binding assay This method is based on depletion of emulsion-bound protein (or peptide) from the infranatant region under conditions where the degree of sedimentation of the unbound protein (or peptide) is negligible The binding profile for apoE(263–286) to Myr2Gro-PCho/TO emulsion fraction is presented in Fig As the peptide concentration is increased, larger amounts of apoE(263–286) bind to the emulsion particles (Fig 5), which is consistent with the results of the flotation velocity analysis (Fig 4A) The shape of the binding profile indicates that saturation is approached at a peptide concentration of 120 lgỈmL)1 (% 50 lM) By plotting the free protein, Pf, against the phospholipid concentration multiplied by the ratio of free to bound peptide, a linear plot results (Fig 5, inset), yielding an apparent dissociation constant, Kd, of 75 lM and binding capacity, Bmax, of approximately four amino acids per phospholipid (Table 2) Interaction of ApoE3 and ApoE4 with Myr2Gro-PCho/TO lipid emulsions To compare the interactions of apoE3 and apoE4 isoforms with size-fractionated lipid emulsions, flotation velocity experiments were conducted in the analytical ultracentrifuge Fraction was employed as a synthetic model for small lipoprotein particles and fraction for large lipoprotein particles The c(sf) distributions of fraction in the absence and presence of 1.0 lM apoE3 or apoE4 are compared in Fig 6A Relative to the control, the c(sf) distribution of fraction in the presence of 1.0 lM apoE3 shows an increase in area under the curve and a shift to lower flotation coefficients, indicating significant amounts of apoE3 bind these small emulsions (Fig 6A) In contrast, the c(sf) distribution of fraction in the presence of 1.0 lM apoE4 is more similar to the control, indicating lesser amounts of apoE4 bind the small emulsion particles As for apoE(263–286), Monte-Carlo analysis demonstrates Ó FEBS 2002 Binding of apolipoprotein E3 and E4 to emulsions (Eur J Biochem 269) 5945 Table Parameters for the binding of apoE(263–286), apoE3 and apoE4 to Myr2Gro-PCho/TO emulsion fraction Kd and Bmax values were calculated according to Eqn (9) Bmax Kd ApoE Isoform or Peptide (lgỈmL)1) (lM) ApoE/particle PL/ApoE Amino acids/PL ApoE(263–286) ApoE3 ApoE4 210 15 17 75 0.44 0.51 3.14 · 104 1010 1630 5.9 163 101 4.1 1.83 2.96 that the observed changes in the c(sf) distributions of fraction in the presence of apoE3 or apoE4 are statistically significant, and cannot be attributed to noise affecting the data analysis (Fig 6B) Analysis of the c(sf) distributions for large emulsions (fraction 6) in the presence and absence of 0.5, 1.0 and 2.0 lM apoE3 or apoE4 reveals an opposite trend (Fig 7A,B) Although there is evidence that apoE3 interacts with fraction 6, given by the shift in the distribution to lower flotation coefficients (Fig 7A), there is a significantly greater increase in the ordinate maximum value and area under the curve for the c(sf) distribution in the presence of apoE4 at corresponding protein concentrations (Fig 7B) This suggests that a higher proportion of apoE4 binds to the large emulsion particles As for fraction (Fig 6B), MonteCarlo analysis of all data sets presented in Fig 7A and B demonstrates that the comparative differences observed for apoE3 and apoE4 are statistically significant (data not shown) fraction in the presence of 1.0 lM apoE4 is similar to the control, suggesting little apoE4 is bound to the fraction particles Likewise, the interaction of apoE3 and apoE4 isoforms with large EggPtdCho/TO emulsions was examined Figure 7C shows the flotation velocity data of EggPtdCho/ TO fraction in the presence and absence of 1.0 lM apoE3 or apoE4 Consistent with earlier observations (Fig 7A,B), a minor shift to smaller flotation coefficients is observed for EggPtdCho/TO fraction in the presence of both apoE isoforms However, a greater increase in the ordinate maximum and area under the curve is obvious in the presence of the apoE4 isoform (Fig 7C, open symbols), indicating that a greater proportion of apoE4 binds the larger particles Together with the results obtained in the previous section employing Myr2Gro-PCho/TO emulsions, these data support the conclusion that apoE3 and apoE4 bind preferentially to small and large lipid particles, respectively Interaction of ApoE3 and ApoE4 with EggPtdCho/TO lipid emulsions Direct binding analysis of ApoE3 and ApoE4 to large emulsion particles The binding of apoE3 and apoE4 to lipid particles was also assessed using size-fractionated emulsions comprised of egg yolk phosphatidylcholine (EggPtdCho) and TO EggPtdCho is comprised of saturated and unsaturated phospholipids, which also differ in fatty acyl chain length, and are therefore more biologically relevant than monolayers comprised of Myr2Gro-PCho alone [46] The EggPtdCho/TO emulsions were synthesized by pressure extrusion and fractionated by sucrose-gradient ultracentrifugation, employing identical procedures to those used for the synthesis of Myr2Gro-PCho/TO emulsions Following fractionation, flotation velocity experiments were performed to characterize the solution properties of these emulsions, which are summarized in Table The data presented in Table demonstrates that the EggPtdCho/TO emulsions share similar physical properties to the fractionated Myr2Gro-PCho/TO emulsions (Table 1) Accordingly, flotation velocity experiments were employed to compare the binding of apoE3 and apoE4 to small (fraction 2) and large (fraction 6) EggPtdCho/TO emulsions As for Myr2Gro-PCho/TO emulsions (Figs 6A,B and 7A,B), identical size-dependent binding preferences were observed (Figs 6C and 7C) Relative to the emulsion sample alone, the flotation rate of fraction in the presence of 1.0 lM apoE3 is significantly decreased, particularly evident for particles with sf < 200 S (Fig 6C), indicating an appreciable amount of apoE3 is bound to the small lipid particles In comparison, the c(sf) distribution of EggPtdCho/TO To verify and quantify the size-dependent interaction of apoE3 and apoE4 to fractionated lipid emulsions, we also employed a direct binding using Myr2Gro-PCho/TO fraction and physiological concentrations of apoE The binding isotherms for the apoE3 and apoE4 isoforms are presented in Fig 8A Both curves show evidence of saturation, although the proportion of bound apoE4 is significantly greater at all protein concentrations employed, particularly in excess of 30 lgỈmL)1 or 1.0 lM (Fig 8A) The apparent Kd and Bmax values for apoE3 and apoE4, calculated from the linearized plots according to Eqn (4) (Fig 8B), are presented in Table In general, these values agree well with previous studies employing purified apoE [2,6] However, the results of these analyses indicate that apoE3 and apoE4, though possessing a similar apparent binding affinity (Kd) for the large lipid particles, are notably distinct in their maximum binding capacities (Table 2) In particular, the binding footprint for the apoE4 isoform, corresponding to 3.0 amino acids per phospholipid, is almost twofold greater than the apoE3 isoform (Table 2), suggesting these isoforms differ in their conformational state when bound to large synthetic emulsions DISCUSSION Besides size, native lipoproteins differ in density, lipid dynamics, lipid composition and apolipoprotein content 5946 M A Perugini et al (Eur J Biochem 269) Fig Flotation velocity analysis of Myr2Gro-PCho/TO and EggPtdCho/TO emulsion fraction in the absence and presence of apoE3 and apoE4 isoforms The c(sf) distribution calculated using an invariant D ¼ 0.63 · 10)7 cm2Ỉs)1 is plotted as a function of flotation coefficient (A) Myr2Gro-PCho/TO fraction alone (solid line, no symbols), and Myr2Gro-PCho/TO fraction in the presence of 1.0 lM apoE3 (solid line, solid symbols) and 1.0 lM apoE4 (solid line, open symbols) Total lipid concentration in Myr2Gro-PCho/TO fraction ¼ 410 lM, i.e [Myr2Gro-PCho] ¼ 150 lM + [TO] ¼ 260 lM (B) Results of Monte-Carlo statistical analysis distributions, calculated from 1000 synthetic data sets to a confidence level of P ¼ 0.95 The lower (0.025) and upper (0.975) quantiles are depicted as dashed lines, enclosing the mean distribution (solid lines) for Myr2Gro-PCho/TO fraction alone (labelled 1), Myr2Gro-PCho/TO fraction + 1.0 lM apoE3 (labelled 2), and Myr2Gro-PCho/TO fraction + 1.0 lM apoE4 (labelled 3) (C) EggPtdCho/TO fraction alone (solid line, no symbols); EggPtdCho/TO fraction + 1.0 lM apoE3 (solid symbols + line) and EggPtdCho/TO fraction + 1.0 lM apoE4 (open symbols + line) The total lipid concentration in EggPtdCho/TO fraction ¼ 280 lM, i.e [EggPtdCho] ¼ 135 lM + [TO] ¼ 145 lM Ĩ FEBS 2002 Fig c(sf) distribution analysis of Myr2Gro-PCho/TO and EggPtdCho/TO fraction in the presence and absence of apoE3 and apoE4 The c(sf) distributions calculated using an invariant D ¼ 0.38 · 10)7 cm2Ỉs)1 are plotted as a function of flotation coefficient (A) Myr2Gro-PCho/TO fraction alone (solid line, no symbols); Myr2Gro-PCho/TO fraction + 0.5 lM apoE3 (dashed line), Myr2Gro-PCho/TO fraction + 1.0 lM apoE3 (dashed-dotted line) and Myr2Gro-PCho/TO fraction + 2.0 lM apoE3 (solid line + open symbols) The total lipid concentration in Myr2Gro-PCho/TO fraction ¼ 125 lM, i.e [Myr2Gro-PCho] ¼ 23 lM + [TO] ¼ 102 lM (B) Myr2Gro-PCho/TO fraction alone (solid line, no symbols); Myr2Gro-PCho/TO fraction + 0.5 lM apoE4 (dashed line), Myr2Gro-PCho/TO fraction + 1.0 lM apoE4 (dashed-dotted line) and Myr2Gro-PCho/TO fraction + 2.0 lM apoE4 (solid line + open symbols) (C) EggPtdCho/TO fraction alone (solid line, no symbols); EggPtdCho/TO fraction + 1.0 lM apoE3 (solid symbols + line) and EggPtdCho/TO fraction + 1.0 lM apoE4 (open symbols + line) The total lipid concentration in EggPtdCho/TO fraction ¼ 94 lM, i.e [EggPtdCho] ¼ 21 lM + [TO] ¼ 73 lM Ĩ FEBS 2002 Binding of apolipoprotein E3 and E4 to emulsions (Eur J Biochem 269) 5947 Table Hydrodynamic properties of EggPtdCho/TO size-fractionated lipid emulsions Lipid emulsions composed of EggPtdCho and TO were fractionated and characterized by size-distribution analysis as described in Experimental procedures The symbols used are sf, flotation coefficient taken from the ordinate maximum of the best-fit c(sf) distribution; M, molar mass taken from the ordinate maximum of the best-fit c(M)  distribution employing Eqn (3); v, partial specific volume calculated from M using Eqn (3) (as above); Rs, particle radii, assuming a spherical  particle and using the experimentally determined values for M and v Fraction # TO:EggPtdCho molar ratio sf (S) M (· 108 Da)  v (mLỈg)1) RS (nm) 1.07 1.66 2.18 2.84 3.54 133 313 487 649 806 0.76 2.31 4.05 5.80 7.60 1.038 1.049 1.056 1.061 1.067 31 46 55 62 68 Fig Binding of apoE3 and apoE4 to Myr2Gro-PCho/TO emulsion fraction (A) The amount of bound apoE3 (solid symbols + solid line) and apoE4 (open symbols + solid line) is plotted as a function of free protein (lgỈmL)1) The concentration of Myr2Gro-PCho and TO in lipid emulsion fraction ¼ 150 lM and 260 lM, respectively Binding data was obtained by analytical ultracentrifugation using the direct binding assay as described in Experimental procedures (B) Linearized plot of the binding data for apoE3 (solid symbols) and apoE4 (open symbols) shown in panel A The solid line represents the linear least-squares fits to the data according to Eqn (4), where the y-intercept and slope equate to the apparent Kd and Bmax, respectively (Table 2) [47–50] This raises the possibility that any one or a combination of these properties may influence the binding of apolipoproteins to lipoprotein particles The capacity to fractionate lipid emulsions into relatively homogeneous particles (Figs and 3) provides an experimental system to directly examine the effect of particle size on apolipoprotein binding Nevertheless, synthetic emulsions can differ markedly in monolayer-core lipid dynamics depending on their lipid composition [51,52] Accordingly, we have employed two different phospholipid-stabilized emulsions to demonstrate that apoE3 and apoE4 discriminate between synthetic lipid emulsions on the basis of size or surface curvature The apoE3 isoform is shown to bind preferentially to small, highly curved lipid emulsions (Fig 6); whereas the apoE4 molecule binds preferentially to large, less curved lipid particles (Fig 7) This behaviour is consistent with previous studies showing that apoE3 and apoE4 distribute preferentially with HDL and VLDL, respectively [7–10] Furthermore, these results may be considered in relation to a number of in vivo observations The elevated LDL concentrations reported in subjects with homozygous E4/4 phenotypes [18] may be due to the inability of apoE4 to bind and initiate the clearance of small lipoproteins in plasma In contrast, the observation that chylomicron remnants are cleared faster in subjects with apoE4, compared to those with apoE3 [53], may be explained by the superior ability of apoE4 to bind large lipid particles Similarly, a sizedependent binding phenomenon may account for the observation that apoE3 distributes preferentially with small (density > 1.125 gỈmL)1) and apoE4 with large (density < 1.00 gỈmL)1) lipoproteins in the cerebrospinal fluid of the brain [54] Insight into the structural basis for apoE3 and apoE4 lipid binding preferences is provided by earlier studies, where it is demonstrated that apoE3 and apoE4 differ in their NH2- and COOH-terminal domain interactions [7,8] In addition, it is known that truncation of apoE4 at residue 244 abolishes VLDL binding [7], although the same truncated variant of apoE3 retains the ability to bind HDL [23] This suggests the important determinants for binding large lipid particles are downstream of residue 244, in the COOH-terminal region of the protein More recently, studies employing fluorescence resonance energy transfer [24,26], intradomain disulfide bonding [3], nuclear magnetic resonance [25], and microcalorimetry [28] experiments demonstrate that the NH2-terminal domain of apoE can reorganize from a closed to open conformation in the Ó FEBS 2002 5948 M A Perugini et al (Eur J Biochem 269) REFERENCES Fig A model for the lipoprotein-bound conformations of human apolipoprotein E isoforms The COOH-terminal domain is shown bound to the lipoprotein surface, whilst the NH2-terminal domain undergoes conformational changes from (A) the closed conformation, to (B) the open conformation Model adapted from [27,28] presence of lipid In view of these observations, differences in the propensity for apoE3 and apoE4 to adopt different conformations on the surface of lipid particles may explain their ability to distribute preferentially to different sized particles The data presented in this study supports this suggestion We used a direct binding assay in the analytical ultracentrifuge to show that apoE3 and apoE4 bind with similar affinity to large lipid emulsions (Kd % 0.5 lM), but differ by almost twofold in their maximum binding capacities (Fig 8, Table 2) The Bmax for apoE4 is approximately 3.0 amino acids per phospholipid, whereas the Bmax for apoE3 is approximately 1.8 amino acids per phospholipid This indicates that apoE4 has a smaller binding footprint when bound to large lipid particles One explanation for this phenomenon could be that apoE4 selfassociates more readily on the surface of lipid emulsions However, we have previously demonstrated that both apoE3 and apoE4 dissociate to folded monomers when complexed with phospholipid micelles [4], consistent also with earlier studies [55,56] Accordingly, we propose that apoE3 and apoE4 differ in their capacity to adopt expanded and compact conformations when bound to lipid particles We suggest that the apoE4 isoform binds more extensively to large, less curved lipid particles due its 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apolipoproteins to lipoprotein particles The capacity to fractionate lipid emulsions into relatively homogeneous