Title: Structural and Functional Variations in Human Apolipoprotein E3 and E4
Abstract: There are three major apolipoprotein E (apoE) isoforms. Although APOE-ϵ3 is considered a longevity gene, APOE-ϵ4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease. There are three major apolipoprotein E (apoE) isoforms. Although APOE-ϵ3 is considered a longevity gene, APOE-ϵ4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease. Human apolipoprotein E (apoE) 2The abbreviations used are: apoE, apolipoprotein E; AD, Alzheimer disease; Aβ, β-amyloid peptide; DMEM, Dulbecco's modified Eagle's medium; DHPC, dihexanoylphosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; HepG2, hepatoblastoma cells; LDL, low density lipoprotein; LDL-R, LDL receptor; VLDL, very low density lipoprotein; Meq, equivalent molar mass; mLV, multilamellar vesicles; PBS, phosphate-buffered saline; r.m.s.d., root mean square deviation; SV, sedimentation velocity; SE, sedimentation equilibrium. 2The abbreviations used are: apoE, apolipoprotein E; AD, Alzheimer disease; Aβ, β-amyloid peptide; DMEM, Dulbecco's modified Eagle's medium; DHPC, dihexanoylphosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; HepG2, hepatoblastoma cells; LDL, low density lipoprotein; LDL-R, LDL receptor; VLDL, very low density lipoprotein; Meq, equivalent molar mass; mLV, multilamellar vesicles; PBS, phosphate-buffered saline; r.m.s.d., root mean square deviation; SV, sedimentation velocity; SE, sedimentation equilibrium. is a 299-amino acid protein with a molecular mass of 34 kDa. ApoE is encoded by the three alleles (APOE-ϵ2, APOE-ϵ3, and APOE-ϵ4) of a gene on chromosome 19q13.2 determining the three major isoforms, namely apoE2, apoE3, and apoE4 in six phenotypes (1Mahley R.W. Rall Jr., S.C. Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1290) Google Scholar). The three apoE isoforms differ from each other only by a single amino acid substitution involving cysteine-arginine replacement at residues 112 and 158, i.e. apoE2 (Cys112/Cys158), apoE3 (Cys112/Arg158), and apoE4 (Arg112/Arg158) (Fig. 1A). Genetically, the APOE-ϵ4 allele is associated with both familial late-onset and sporadic Alzheimer disease (AD) and atherosclerosis (2Greenow K. Pearce N.J. Ramji D.P. J. Mol. Med. 2005; 83: 329-342Crossref PubMed Scopus (183) Google Scholar, 3Lane R.M. Farlow M.R. J. Lipid Res. 2005; 46: 949-968Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 4Tanzi R.E. Bertram L. Cell. 2005; 120: 545-555Abstract Full Text Full Text PDF PubMed Scopus (1458) Google Scholar). AD patients carrying the APOE-ϵ4 allele have more profound deposition of β-amyloid peptides (Aβ) in their brains than those carrying APOE-ϵ2 and APOE-ϵ3 alleles (5de la Torre J.C. J. Alzheimers Dis. 2002; 4: 497-512Crossref PubMed Scopus (91) Google Scholar, 6Farrer L.A. Cupples L.A. Haines J.L. Hyman B. Kukull W.A. Mayeux R. Myers R.H. Pericak-Vance M.A. Risch N. van Duijn C.M. J. Am. Med. Assoc. 1997; 278: 1349-1356Crossref PubMed Google Scholar). Considerable evidence supports the view that apoE4 increases the risk of AD by accelerating the plaque formation and by impairing the neurons. ApoE4 appears to modulate amyloid precursor protein processing and Aβ production through both the LDL receptor-related protein pathway and domain interaction (7Ye S. Huang Y. Mullendorff K. Dong L. Giedt G. Meng E.C. Cohen F.E. Kuntz I.D. Weisgraber K.H. Mahley R.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18700-18705Crossref PubMed Scopus (211) Google Scholar). Strong correlation of APOE-ϵ4 allele with dyslipidemia and atherosclerosis, the major underlying mechanism of coronary heart disease, has been demonstrated (8Gregg R.E. Brewer Jr., H.B. Clin. Chem. 1988; 34: B28-B32PubMed Google Scholar). Human apoE4 represents a dual risk factor for these two major degenerative diseases. ApoE contains two independently folded domains (N-terminal domain, residues 20-165, and C-terminal domain, residues 225-299) that are separated by a large nonstructural segment (9Nolte R.T. Atkinson D. Biophys. J. 1992; 63: 1221-1239Abstract Full Text PDF PubMed Scopus (153) Google Scholar) (Fig. 1A). The four helices in the N-terminal domain are amphipathic (10Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). The three-dimensional structures of the N-terminal domain of apoE isoforms have been determined by x-ray crystallography (11Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (593) Google Scholar, 12Dong L.M. Wilson C. Wardell M.R. Simmons T. Mahley R.W. Weisgraber K.H. Agard D.A. J. Biol. Chem. 1994; 269: 22358-22365Abstract Full Text PDF PubMed Google Scholar, 13Wilson C. Mau T. Weisgraber K.H. Wardell M.R. Mahley R.W. Agard D.A. Structure. 1994; 2: 713-718Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), which showed that this domain is arranged as an anti-parallel, elongated four-helix bundle. The C-terminal domain, which is predicted to contain three helices, is less well known. The first two helices, comprising residues 203-223 and residues 225-266, are class A type, and the third helix, consisting of residues 268 to 289, is a G* helix (9Nolte R.T. Atkinson D. Biophys. J. 1992; 63: 1221-1239Abstract Full Text PDF PubMed Scopus (153) Google Scholar). The G* helix and part of the end of the second helix may play a key role in lipid binding and lipid interaction in apoE-containing lipoproteins (14Sparrow J.T. Sparrow D.A. Fernando G. Culwell A.R. Kovar M. Gotto Jr., A.M. Biochemistry. 1992; 31: 1065-1068Crossref PubMed Scopus (42) Google Scholar). In the lipid-free state, full-length apoE exists as a stable tetramer, and the C-terminal domain is responsible for tetramerization (15Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C.S. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar). ApoE4 is one of the major proteins associated with Aβ plaques (16Wisniewski T. Castano E.M. Golabek A. Vogel T. Frangione B. Am. J. Pathol. 1994; 145: 1030-1035PubMed Google Scholar, 17Puglielli L. Tanzi R.E. Kovacs D.M. Nat. Neurosci. 2003; 6: 345-351Crossref PubMed Scopus (686) Google Scholar). The role of apoE in the molecular pathogenesis of AD might be related to its isoform-specific interactions with lipids or Aβ aggregates. The N terminus of apoE, containing a region spanning from residues 140 to 150 (His-Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg), is rich in basic amino acids and is involved in apoB/E receptor binding (18Innerarity T.L. Friedlander E.J. Rall S.C. Jr-Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1983; 258: 12341-12347Abstract Full Text PDF PubMed Google Scholar, 19Dyer C.A. Curtiss L.K. J. Biol. Chem. 1991; 266: 22803-22806Abstract Full Text PDF PubMed Google Scholar, 20Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Most of the basic amino acids in the vicinity of residues 136-158 are not involved in salt bridges and are on the solvent-accessible surface (13Wilson C. Mau T. Weisgraber K.H. Wardell M.R. Mahley R.W. Agard D.A. Structure. 1994; 2: 713-718Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). ApoE in VLDL promotes the catabolism of VLDL to LDL (8Gregg R.E. Brewer Jr., H.B. Clin. Chem. 1988; 34: B28-B32PubMed Google Scholar). ApoE-deficient (apoE(-)) mice exhibit elevated plasma cholesterol because of impaired VLDL metabolism and develop atherosclerosis spontaneously (21Palinski W. Ord V.A. Plump A.S. Breslow J.L. Steinberg D. Witztum J.L. Arterioscler. Thromb. 1994; 14: 605-616Crossref PubMed Scopus (471) Google Scholar). To correlate the structural variation between apoE4 and apoE3 isoforms and their functional disturbances, we have expressed full-length, N-terminal, and C-terminal truncated apoE proteins tailored to interrupt the potential domain or segment interactions (22Chou C.Y. Lin Y.L. Huang Y.C. Sheu S.Y. Lin T.H. Tsay H.J. Chang G.G. Shiao M.S. Biophys. J. 2005; 88: 455-466Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Using analytical ultracentrifugation coupled with continuous size distribution analysis, we have demonstrated previously that apoE3-(72-299) consists of only one major species (with a sedimentation coefficient of 5.1). ApoE4-(72-299) displays a wider and more complicated species distribution. The two major species of monomer and tetramer distribution are maintained in the case of apoE3-(1-231) and apoE4-(1-231). In this study, we further elucidate the de-aggregation of different apoE3 and apoE4 protein in the presence of dihexanoylphosphatidylcholine (DHPC). DHPC has the chemical structure of phospholipids and exists in solution as monomers in equilibrium with micelles comprising ∼40 molecules (the critical micelle concentration is 12.7-14.3 mm) (23Tausk R.J. Karmiggelt J. Oudshoorn C. Overbeek J.T. Biophys. Chem. 1974; 1: 175-183Crossref PubMed Scopus (184) Google Scholar, 24Burns R.A. Jr-Roberts M.F. Biochemistry. 1980; 19: 3100-3106Crossref PubMed Scopus (61) Google Scholar). Besides sedimentation velocity, sedimentation equilibrium experiments were also used to calculate the equivalent molar mass of different apoE3 and apoE4 proteins in PBS and in the presence of lipid. Together, global analysis of both sedimentation velocity and sedimentation equilibrium data gave very reliable results on the aggregational states of apoE proteins in different solvent systems. Furthermore, we report the functional studies of these isoform-specific, domain-truncated apoE proteins, including the interaction with lipid vesicles, binding with apoB/E receptor in HepG2 cells, and the cholesterol-lowering effects in apoE(-) mice. Our results provide novel evidence to correlate the structure variation of apoE3 and apoE4 to their biochemical functions and possible pathophysiologic consequences in atherosclerosis and AD. Plasmids—The pET-29a(+) (Novagen) vectors with a C-terminal His tag sequence (Ser2-His6) were used. The construction of pET-apoE3, pET-apoE3-(41-299), pET-apoE3-(72-299), pET-apoE3-(1-191), pET-apoE3-(1-231), pET-apoE3-(1-271), and those of apoE4 vectors was described previously (22Chou C.Y. Lin Y.L. Huang Y.C. Sheu S.Y. Lin T.H. Tsay H.J. Chang G.G. Shiao M.S. Biophys. J. 2005; 88: 455-466Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The human apoE3-(72-166) cDNA fragments were amplified by PCR, and the forward primer was 5′-AAACATATGAAGGCCTACAAATCGGA, whereas the reverse primer was 5′-AACTCGAGGGCCCCGGCCT. The NdeI-XhoI-digested apoE3-(72-166) cDNA was then ligated to the 5.2-kb NdeI-XhoI fragment from pET-29a(+). In turn, this resulted in a 5.5-kb pET-apoE3-(72-166) vector. Site-directed mutagenesis (25Braman J. Papworth C. Greener A. Methods Mol. Biol. 1996; 57: 31-44PubMed Google Scholar) was used to construct pET-apoE4-(72-166). The forward primer was 5′-GAGGACGTGCGCGGCCGCCTG and the reverse primer was 5′-AGGCGGCCGCGCACGTCCTCC. The pET-apoE3-(72-166) vectors were used as templates, and the primers were used to mutate the Cys112 codon to Arg112 codon for apoE4 by PCR. Their nucleotide sequences were checked by autosequencing analysis. Purification of the Full-length, N-terminal, and C-terminal Truncated ApoE Proteins—The procedure of protein induction and purification was described previously (22Chou C.Y. Lin Y.L. Huang Y.C. Sheu S.Y. Lin T.H. Tsay H.J. Chang G.G. Shiao M.S. Biophys. J. 2005; 88: 455-466Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The BL21-CodonPlus(DE3)-RIPL Escherichia coli cells (Stratagene) were used for the protein expression of apoE3 and apoE4 proteins. The others were expressed by the strain of BL21 (DE3) (Invitrogen). Typical yields of the apoE3 and apoE4 proteins were 5 mg and those of apoE3 and apoE4 N- or C-terminal truncated proteins were 5-20 mg after purification from 1 liter of E. coli culture medium. The purified proteins were buffer-changed by Amicon Ultra-4 centrifugal filter devices (Millipore) with the molecular mass cutoff at 10 kDa. After repeating the concentration-dilution procedure five times, the elution buffer had been fully replaced by PBS (pH 7.3). Sedimentation Velocity—Sedimentation velocity (SV) experiment was performed using a Beckman model XL-A analytical ultracentrifuge (Fullerton, CA). Briefly, samples (380 μl) and reference (400 μl) solutions were loaded into 12-mm double-sector Epon charcoal-filled centerpieces and mounted in an An-50 Ti rotor. Experiments were performed at 20 °C with a rotor speed of 42,000 rpm. Absorbance of the sample at 280 nm was monitored in a continuous mode time interval of 360-480 s and a step size of 0.003 cm. Multiple scans at different time points were fitted to a continuous size distribution model by using the SEDFIT (26Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (2978) Google Scholar, 27Schuck P. Perugini M.A. Gonzales N.R. Howlett G.J. Schubert D. Biophys. J. 2002; 82: 1096-1111Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar) program as described previously (22Chou C.Y. Lin Y.L. Huang Y.C. Sheu S.Y. Lin T.H. Tsay H.J. Chang G.G. Shiao M.S. Biophys. J. 2005; 88: 455-466Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). All size distributions were solved and regularized on a confidence level of p = 0.68 (Fig. 1, B-D) or 0.95 (Fig. 2) by maximum entropy, the best fitted average anhydrous frictional ratio (f/f0), and a resolution N of 200 sedimentation coefficients between 0.1 and 20.0 S. Sedimentation Equilibrium—Experiments were performed with six-channel Epon charcoal-filled centerpieces. Three different samples (0.10-0.12 ml) were loaded into the sample channels, and 0.11-0.13-ml buffers only were loaded into the reference channels. The cells were then loaded into the rotor and run at speed of 6,000, 10,000, 15,000, and 20,000 rpm each for 14-18 h at 20 °C. Ten A280 nm scans with time interval of 8-10 min were measured for every different rotor speed to check the status of sedimentation equilibrium (SE). In our studies, all apoE proteins can achieve equilibrium state after 14 h. The scans at different rotor speeds (multispeed equilibrium data) were then globally fitted to a noninteracting discrete species model assuming a single species by using SEDPHAT (28Vistica J. Dam J. Balbo A. Yikilmaz E. Mariuzza R.A. Rouault T.A. Schuck P. Anal. Biochem. 2004; 326: 234-256Crossref PubMed Scopus (307) Google Scholar) with Equation 1, AR=cr0ϵdexpM(1-ν¯ρ)ω22RT(r2-r02)(Eq. 1) in which r denotes the distance from center of rotation; r0 is an arbitrary reference radius; ω is the angular velocity; T is the absolute temperature of the rotor; R is the gas constant; ν¯ is the partial specific volume; ρ is the solvent density; ϵ is the extinction coefficient; d is the optical path length, and cr0 is the concentration at the reference radius. At each channel, a single base-line parameter was included as a floating parameter common to all rotor speeds. The time invariant and radial invariant noise were also fitted for the better fitting quality. Global analyses of combined sedimentation equilibrium and sedimentation velocity data were conducted with SEDPHAT using hybrid local continuous distribution and global discrete species model (29Schuck P. Scott D.J. Harding S.E. Rowe A.J. Modern Analytical Ultracentrifugation: Techniques and Methods. Royal Society of Chemistry, Cambridge, UK2005: 26-50Google Scholar) with Equation 2, a(r,t)≅∑iC(i)c1(si,Mi,r,t)+∑j∫smin,jsmax,jc(j)(s)c1(s,(f/f0)w,j,r,t)ds(Eq. 2) in which cj(s) denoting the population of species with sedimentation coefficients in different nonoverlapping intervals Ij = |smin,j, smax,j| that can be characterized by separate frictional ratios, in combination with discrete species at loading concentration ci (in signal units) with s values si outside the intervals Ij. The discrete species are described by Lamm equation solutions, each with the two parameters s and M, and are not connected to the determination of (f/f0)w,j for the continuous segments. In our studies, the resolution of the local c(s) was set to zero to switch it off. Only multiple discrete species analysis was used. DMPC Turbidity Clearance Assay—Dimyristoylphosphatidylcholine (DMPC), purchased from Sigma, was dissolved in a 2:1 (v/v) chloroform/methanol solution. The solvent was evaporated overnight under a nitrogen stream to form a thin film on the walls of a 13 × 100-mm glass tube. The clearance buffer (PBS with 3.5% KBr and 0.1 mm EDTA) was then added into the tube and vortexed vigorously for 2 min. Dilution to 0.5 mg/ml resulted in a turbid suspension of multilamellar vesicles (mLV) with an absorbance of ∼0.9 at 325 nm. The solubilization of DMPC mLV by apoE proteins was determined according to modified procedures (30Li X. Kypreos K. Zanni E.E. Zannis V. Biochemistry. 2003; 42: 10406-10417Crossref PubMed Scopus (47) Google Scholar, 31Pownall H.J. Massey J.B. Kusserow S.K. Gotto Jr., A.M. Biochemistry. 1978; 17: 1183-1188Crossref PubMed Scopus (187) Google Scholar, 32Segall M.L. Dhanasekaran P. Baldwin F. Anantharamaiah G.M. Weisgraber K.H. Phillips M.C. Lund-Katz S. J. Lipid Res. 2002; 43: 1688-1700Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Briefly, apoE (250 μg) was added to DMPC mLV solution (0.5 mg/ml) in a quartz cuvette, which was preincubated at 24 °C in a spectrophotometer (PerkinElmer Life Sciences, Lambda 35) with water-circulated temperature control. The contents were mixed within 10 s by repeated pipetting. The vesicle solubilization was monitored as a decrease in absorbance at 325 nm. Experiments were repeated at least twice, and similar results were obtained. According to Segall et al. (32Segall M.L. Dhanasekaran P. Baldwin F. Anantharamaiah G.M. Weisgraber K.H. Phillips M.C. Lund-Katz S. J. Lipid Res. 2002; 43: 1688-1700Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), the time courses for clearance were fitted by nonlinear regression to the monoexponential Equation 3, Y=A·e-k·t+C(Eq. 3) or the biexponential Equation 4, Y=A·e-k1·t+B·e-k2·t+C(Eq. 4) where Y is the absorbance at 325 nm, and k, k1, or k2 are the rate constants for different kinetic phases of the solution clearance. A and B are the changes in turbidity for different phases (pool sizes); t is time, and C is the remaining turbidity at the completion of the reaction. Slight differences in the initial absorbance for each time course are corrected by normalizing these values to 1 and treating all others as the fractions of this initial turbidity. Preparation of 3H-LDL—Unlabeled cholesterol (0.1 mg, dissolved in 1.0 ml of n-hexane) was added to a clean glass vial and dried under a stream of nitrogen to form a thin cholesterol film on the surface of 2-cm-high wall in a glass vial. [7-3H]Cholesterol (125-250 μCi in ethanol) (Amersham Biosciences) was added and dried under nitrogen. Human LDL (1.4 mg/ml protein) of 1-2 ml was added and incubated at 37 °C for 22 h. Finally, the 3H-LDL was centrifuged at 12,000 rpm for 5 min. The upper layer and trace insoluble in the bottom were discarded. The middle clear solution containing 3H-LDL was transferred into a clean tube and sealed under nitrogen. Radioactivity of a small portion of 3H-LDL (10 μl) was determined by a liquid scintillation counter (Beckman). LDL Receptor Binding Assay—The procedure was modified from previous studies (33Krempler F. Kostner G.M. Friedl W. Paulweber B. Bauer H. Sandhofer F. J. Clin. Investig. 1987; 80: 401-408Crossref PubMed Scopus (27) Google Scholar, 34Lundberg B.B. Suominen L.A. Biochem. J. 1985; 228: 219-225Crossref PubMed Scopus (15) Google Scholar). Human hepatoblastoma cells (HepG2) (1.5 × 105) were cultured into a 1-cm diameter well in DMEM with 10% fetal bovine serum at 37 °C with 5% CO2. After a 48-h incubation, cells of 50-60% confluence were washed three times with PBS and then incubated in the serum-free medium for 24 h at 37 °C with 5% CO2. When the assay began, the cells were cooled on ice for 30 min, washed twice with PBS, and then incubated with DMEM containing 50 μg/ml 3H-LDL, and different receptor-binding competitors (apoE) at 4 °C for 2 h. The medium was removed followed by washing three times with chilled PBS. Cells were released from the well surface by 0.2 ml of trypsin-EDTA, pipetted into a clean vial with 2 ml of 1:3 (v/v) saturated KOH/ethanol solution, and incubated at 56 °C for 2 h. Seven milliliters of n-hexane was then added into the vial for dissolving cholesterol and dried under nitrogen to form a thin film on the vial walls. Finally, every vial was added with 10 ml of Aquasol (PerkinElmer Life Sciences), and the radioactivity was determined. In Vivo Cholesterol-lowering Effect—The ability of apoE proteins to lower plasma cholesterol in vivo was assayed by injection of proteins into apoE(-) mice (Animal Center, National Cheng Kung University, Tainan, Taiwan). Male apoE(-) mice, 6 months old, were maintained in the specific pathogen-free animal house with a 12-h light-dark cycle. The fasting plasma total cholesterol level of each apoE(-) mouse 1 day before apoE protein injection, in the range of 650-750 mg/dl (∼2.88 × 10-5 mol per 20-g mice, whose total blood volume is about 8% of the body weight) (35Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1818) Google Scholar, 36Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer H.B. J. Clin. Investig. 1995; 96: 1612-1620Crossref PubMed Scopus (122) Google Scholar, 37Hoff J. Lab. Anim. 2000; 29: 47-53Google Scholar), was used as its own control. The cholesterol content of VLDL + LDL of an animal was estimated to be about 2.88 × 10-5 mol. For each VLDL particle (∼2000 cholesterol included) to accommodate at least one apoE molecule, the required amounts of injected proteins were estimated to be 490 μg for apoE, 420 μg for apoE-(41-299), 370 μg for apoE-(72-299), and 390 μg for apoE-(1-231). Animals were randomly assigned for each group (n = 6-8) and were injected from tail veins with 200 μl for all protein solution in PBS. By determining the concentration of total cholesterol before and 24 h after injection, the cholesterol-lowering effect was determined. Because the original concentration of cholesterol associated with VLDL + LDL was high and the concentration of high density lipoprotein cholesterol in apoE(-) mice remained approximately constant (about 50 mg/dl) (35Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1818) Google Scholar), the plasma cholesterol-lowering effect was mainly contributed by apoE proteins to accelerate VLDL clearance in the plasma. Self-aggregation of ApoE Proteins Determined by Size Distribution Analysis—ApoE3, apoE4, and their truncated proteins were chosen for this study. Fig. 1A shows the model structure of apoE protein with different colors representing the truncated regions. Our previous study (22Chou C.Y. Lin Y.L. Huang Y.C. Sheu S.Y. Lin T.H. Tsay H.J. Chang G.G. Shiao M.S. Biophys. J. 2005; 88: 455-466Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) has elucidated the structural variation in apoE3 and apoE4 with respect to the N-terminal and C-terminal truncations. In this study, the protein concentration of 0.5 mg/ml was used to make clear that the structural variation of apoE3 and apoE4 is at the same tendency. ApoE3 and apoE4 proteins showed the similar size distribution patterns by continuous c(s) size distribution analysis (Fig. 1B). The root mean square deviation (r.m.s.d.) of the best fitting to the continuous size distribution model was 0.00578 for apoE3 and 0.00551 for apoE4. The grayscale patterns of residual bit maps showed a high quality fitting (Fig. 1, B-D, insets). The best fit f/f0 was 1.21 for apoE3 and 1.28 for apoE4. After calculating the integral area by SEDFIT, apoE3 could be resolved into five major species at s = 2.1 (3%), 4.9 (13%), 6.6 (35%), 8.6 (24%), and 10.8 (13%) and those of apoE4 were s = 1.7 (4%), 4.8 (19%), 6.3 (36%), 7.9 (21%), and 9.6 (9%). Their large components (s > 11), which were lower in content and broader in distribution, were calculated as a single region. ApoE3 and apoE4 had similar percentage of larger species (s > 11), namely 12% for apoE3 and 11% for apoE4. Compared with the simplicity in size distribution of apoE3-(72-299), apoE4-(72-299) existed in a more complicated and aggregated pattern (Fig. 1C). The r.m.s.d. of the best fitting was 0.00440 for apoE3-(72-299) and 0.00453 for apoE4-(72-299). The best fit f/f0 was 1.56 for apoE3-(72-299) and 1.44 for apoE4-(72-299). The species of apoE3-(72-299) appeared at s = 1.9 (11%), 5.2 (66%), 6.7 (21%), and 8.9 (3%), and larger species (s > 10, 1%) whereas those of apoE4-(72-299) appeared at s = 2.2 (4%), 5.4 (25%), 7.1 (35%), 8.9 (17%), and 10.8 (12%), and s > 12.0 (8%). When residues 232-299 were truncated, the two major species (s = 2.2 and 5.0) were maintained for both apoE3-(1-231) and apoE4-(1-231) (Fig. 1D). However, the contents of these two species were different between the two isoforms (apoE3-(1-231), 43% for s = 2.2 and 42% for s = 5.0 versus apoE4-(1-231), 29% for s = 2.0 and 53% for s = 4.9). Both apoE3-(1-231) and apoE4-(1-231) had a minor species at s = 6.9, whose content was 9% for apoE3-(1-231) and 11% for apoE4-(1-231). The r.m.s.d. of the best fitting was 0.00617 for apoE3-(1-231) and 0.00611 for apoE4-(1-231). The f/f0 of the best fit was 1.58 and 1.46 for apoE3-(1-231) and apoE4-(1-231), respectively. Monte-Carlo statistical analysis (supplemental Fig. 1S) demonstrated that the peaks in the size distribution were robust against noise, indicating that they were not because of reported oscillations from noise-induced artifacts in the regularization (26Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (2978) Google Scholar). Dissociation of ApoE Proteins in the Presence of Dihexanoylphosphatidylcholine Micelles—Because apoE proteins are intimately involved in lipid metabolism, it is ultimately important to assess the state of association of various apoE-truncated constructs in the presence of lipids, especially phospholipids. The commonly employed DMPC mLV lipid bilayer model system is too big for sedimentation studies. We chose to use the short acyl chain DHPC micellar system to address this important issue. The distributions calculated for different apoE proteins (initial concentration was 0.5 mg/ml) in the presence of 50 mm DHPC are shown in Fig. 2. All apoE proteins showed significant protein dissociation by DHPC. Each protein appeared as a major species at s = 1.6-2.2 (79, 73, 65, 75, 85, and 64% for apoE3, apoE4, apoE3-(72-299), apoE4-(72-299), apoE3-(1-231), and apoE4-(1-231), respectively). The r.m.s.d. of the best fitting to size distributions were from 0.00645 (apoE3-(72-299)) to 0.00977 (apoE4). Although the residual bit maps (Fig. 2, A-C, insets) showed some minor nonhomogeneous grayscale, the fitting quality was still reliable. In addition, the best fit f/f0 values