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* Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan; Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan; and Faculty of Life Sciences, Institute of Biochemistry, ¶ Institute of Neuroscience, and || Structural Biology Program, National Yang-Ming University, Taipei 112, Taiwan
Correspondence: Address reprint requests to Gu-Gang Chang, Faculty of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan. Tel.: 886-2-2826-7168; Fax: 886-2-2820-2449; E-mail: ggchang{at}ym.edu.tw; or to Ming-Shi Shiao, Dept. of Medical Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan. Tel.: 886-2-2875-1622; Fax: 886-2-2875-1562; E-mail: msshiao{at}vghtpe.gov.tw.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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-helical structure with more hydrophobic residue exposure. The structural variation of the N-terminal truncated apoE3 and apoE4 protein provides useful information that helps to explain the greater aggregation of the apoE4 isoform and thus has implication for the involvement of apoE4 in AD. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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; Selkoe, 2001; Van Bockxmeer et al., 1994; Demant et al., 1991; Kao et al., 1995; Dong et al., 1994; Lalazar and Mahley, 1989). There are three major isoforms of human apoE (namely apoE2, apoE3, and apoE4), which are the products of three alleles (
2, 3, and 4) at a single gene locus on chromosome 19q13.2. The most common isoform of apoE is apoE3, which contains cysteine and arginine at positions 112 and 158. Both positions contain cysteine residues in apoE2 and arginine residues in apoE4 (Fig. 1). Strong correlation of 4 allele with dyslipidemia and coronary heart disease (CHD) has been demonstrated. The estimated CHD odds associated with the 4 allele is greater than that for any other known genetic abnormality. The association of 4 allele with CHD remains significantly high in women, and both sexes combined, after adjustment for traditional coronary risk factors and plasma lipids. ApoE4 homozygosity is also a determinant of restenosis after coronary angioplasty (Van Bockxmeer et al., 1994). ApoE-deficient (apoE(–)) mice develop atherosclerosis spontaneously and the severity is enhanced by a high-cholesterol diet.
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; Schellenberg, 1995; Marx, 1993; Pericak-Vance and Haines, 1995; Strittmatter and Roses, 1995; Polvikoski et al., 1995; Henderson et al., 1995; Huang et al., 2001; Nathan et al., 1994; Sanan et al., 1994; Raffai and Weisgraber, 2003; Chauhan, 2003). The 4 allele is associated with both familial late-onset and sporadic AD, and the gene dose of 4 is a major risk factor for the disease. Accumulated apoE is found in the senile plaques and neurofibrillary tangles (NFT) seen on postmortem examination of the brains of AD patients. AD patients who are homozygous for the 4 allele exhibit more highly developed senile plaques at autopsy. In cultures of dorsal root ganglion neurons, apoE3 increases neurite outgrowth, whereas apoE4 decreases outgrowth (Nathan et al., 1994). ApoE associates with ß-amyloid peptides (Aß) to form novel monofibrils (Sanan et al., 1994). Furthermore, the neuropathology and the apoE profile of aged chimpanzees have implications for AD (Gearing et al., 1994) and C-terminal truncated apoE4 causes AD-like neurodegeneration and behavioral deficits in transgenic mice (Harris et al., 2003).
Recent evidence has indicated that apoE is folded into two independent structural domains (Dong et al., 1994). Limited thrombolytic digestion provides two large fragments with one fragment corresponding to residues 1–199, a 22-kDa N-terminal domain and the other one to residues 216–299, a 10-kDa C-terminal domain (Fig. 1). Crystallographic studies suggest an amphipathic, extended, and antiparallel four-helix bundle for the N-terminal domain (Wilson et al., 1991). The region of residues 136–158, which is rich in basic amino acids, is involved in LDL receptor binding (Siest et al., 1995). The apoE4 isoform binds preferentially to VLDL and the interaction of Arg-61 with Glu-255 may stabilize an extended helical structure in the carboxyl terminus to accommodate a larger, less-curved VLDL surface (Dong and Weisgraber, 1996). The C-terminal domain of apoE beyond residue 191 contains three predicted helices, namely residues 203–223, 225–266, and 268–289 (Wilson et al., 1991; De Pauw et al., 1997). The end of the second helix may play a key role in lipid binding in apoE and lipoprotein interaction (Westerlund and Weisgraber, 1993; Weisgraber, 1994). The third helix (G* helix) induces the aggregation of C-terminus that becomes monomer after the polar/charged mutation of five residues (Phe-257, Trp-264, Val-269, Leu-279, and Val-287; Fan et al., 2004). Choy et al. (2004) have also shown intermolecular coiled-coil helical formation in the C-terminal domain. The C-terminal region of apoE may also interact with Aß in forming NFT (Huang et al., 2001). The N-terminal and C-terminal truncated apoE4 proteins have been found to occur in AD brains and induced intracellular NFT-like inclusions in cultured neurons (Huang et al., 2001). However, the structural characters of apoE isoforms that may lead to the differences in molecular pathogenesis of AD remain poorly understood.
To elucidate the structural difference between apoE3 and apoE4, especially with respect to N-terminal and C-terminal truncation, we have expressed full-length and N- or C-terminal-truncated human apoE3 and apoE4 proteins and compared secondary structure, tertiary structure, and quaternary species distribution.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Construction of human apoE3 expression vectors
The human apoE3 cDNA fragments were first amplified by PCR and ligated into pGEM-T vector (Promega, Madison, WI). The forward primer was 5'-CATATGAAGGTGGAGCAAGCGGTG, and the reverse primer was 5'-GCTCGAGTGATTGTCGCTGGGCACA. The apoE3 cDNA-carried T vectors were then digested with NdeI and XhoI. Finally, the 0.9-kb fragment end product was ligated to the 5.2-kb NdeI-XhoI fragment from pET-29a (+). This in turn resulted in a 6.1-kb pET-apoE3 vector, in which apoE3 cDNA was driven by the T7 promoter and the protein was C-terminally fused to a His·Tag sequence (Ser2-His6).
Construction of N-terminal or C-terminal truncated apoE3 expression vectors
The pET-apoE3 vector was used as the template. The apoE341-299, apoE372-299, apoE31-191, apoE31-231, and apoE31-271 cDNA fragments were amplified by PCR and ligated into pGEM-T vector (Promega). For apoE341-299, the forward primer was 5'-CATATGCAGACACTGTCTGAGCAGG and for apoE372-299, 5'-CATATGAAGGCCTACAAATCGGAACT. The reverse primer for apoE341-299 and apoE372-299 was 5'-CTCGAGGTGATTGTCGCTGGGCACA. The forward primer for apoE31-191, apoE31-231, and apoE31-271 was 5'-TATACATATGAAGGTGGAGCAAG. The reverse primer for apoE31-191 was 5'-AACTCGAGCCGCACGCGGC; for apoE31-231, 5'-AACTCGAGCTCGTCCAGGCGG; and for apoE31-271, 5'-AACTCGAGGTCTTCCACCAGGG. After digesting with NdeI and XhoI, the cDNA fragment end products were ligated to the 5.2-kb NdeI-XhoI fragment from pET-29a (+).
Construction of human apoE4 expression vectors
Site-directed mutagenesis (Braman et al., 1996) was used to construct pET-apoE4, pET-apoE441-299, pET-apoE472-299, pET-apoE41-191, pET-apoE41-231, and pET-apoE41-271 vectors. The forward primer was 5'-GAGGACGTGCGCGGCCGCCTG and the reverse primer was 5'-AGGCGGCCGCGCACGTCCTCC. Briefly, pET-apoE3, pET-apoE341-299, pET-apoE372-299, pET-apoE31-191, pET-apoE31-231, and pET-apoE31-271 vectors were used as templates and the desired primers were used to mutate the Cys-112 codon to the Arg-112 codon by PCR using Pfu DNA polymerase (Promega). After 16–18 temperature cycles, mutated plasmid containing staggered nicks were generated. The PCR products were then treated with DpnI (New England Biolabs, Beverly, MA) to digest the pET-apoE3 templates. Finally, the nicked vector DNA incorporating the desired mutations was transformed into E. coli and their DNA sequence was checked by autosequencing.
Purification of the full-length, N-terminal, and C-terminal truncated apoE proteins
After induced by IPTG at 18°C overnight (apoE full-length, apoE1-191, apoE1-231, and apoE1-271) or 37°C for 3–5 h (apoE41-299 and apoE72-299), the cells were centrifuged at 6000 x g at 4°C for 10 min. The supernatant was removed and the cell pellets were again suspended in the binding buffer (4 mM imidazole, 300 mM sodium chloride, and 20 mM Tris-HCl, pH 7.9). The cells were sonicated by 60 10-s bursts at 300 W with a 10-s cooling period between each burst. The lysate was centrifuged at 10,000 x g for 25 min at 4°C to pellet the cellular debris. One milliliter of the 50% Ni-NTA slurry was equilibrated with the binding buffer and then added to the cleared lysate and the solution mixed gently at 4°C for 60 min. The lysate-Ni-NTA mixture was loaded into a column and washed with the washing buffer (40 mM imidazole, 300 mM sodium chloride, and 20 mM Tris-HCl, pH 7.9). Finally, apoE proteins were eluted with the elution buffer (400 mM imidazole, 300 mM sodium chloride, and 20 mM Tris-HCl, pH 7.9). The purity of all recombinant proteins was estimated to be >95% by the SDS-PAGE. The molar masses of the purified apoE3 and apoE4 proteins (35,365 and 35,418 Da, respectively) were determined by electrospray mass spectrometry and agreed with the theoretical values. The molecular weights of apoE41-299, apoE72-299, apoE1-191, apoE1-231, and apoE1-271, were 30, 27, 23, 28, and 33 kDa, respectively, as anticipated (Fig. 2). Typical yields of the full-length apoE3 and apoE4 proteins were 1.0 mg/l and those of apoE3 and apoE4 N- or C-terminal truncated proteins were 5.0 mg/l after purification.
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Sedimentation velocity
Sedimentation velocity experiments were performed using a Beckman model XL-A analytical ultracentrifuge (Fullerton, CA). Before experiments, samples in PBS buffer were diluted to 0.15, 0.50, or 1.00 mg/ml. Samples (400 µl) and reference (440 µl) solutions were loaded into double-sector centerpieces and mounted in a Beckman An-50 Ti rotor. Experiments were performed at 20°C with a rotor speed of 40,000 rpm. Absorbance of the sample at 280 nm was monitored in a continuous mode, time interval of 480 s, and a step size of 0.002 cm without averaging. Multiple scans at different time points were fitted to a continuous size distribution (see below) by using the program SEDFIT (Schuck, 2000; Schuck et al., 2002) (http://www.analyticalultracentrifugation.com/download.htm) and to a monomer-tetramer rapid self-association model (see below) by using the program SEDPHAT (Schuck, 2003) (http://www.analyticalultracentrifugation.com/sedphat/sedphat.htm). To evaluate the weight-average sedimentation coefficients (sw), the following equation, which was modified from Fujita (1975) and Baldwin (1953), was used (Eq. 1).
 | (1) |
).
Continuous size distribution analysis
The continuous size (mass or sedimentation coefficient) distribution of the proteins can be determined by using finite element solutions of the Lamm equation combined with size distribution analysis techniques by regularization (Schuck, 2000; Schuck et al., 2002). In brief, the observed sedimentation profiles are described as a superimposition of the contributions of each subpopulation c(s) of particles with sedimentation coefficients between s and s + ds by the integral equation (Eq. 2).
 | (2) |
). Similarly, the size distribution can be calculated as a molar mass distribution, c(M), according to Eq. 3.
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The distributions were calculated using maximum entropy regularization, selecting the most parsimonious distribution within a predefined confidence level p for the quality of fit. An average anhydrous ratio (f/f0) representing the ratio of frictional coefficient to that of an anhydrous sphere was employed, together with the Stokes-Einstein and the Svedberg equation, for estimating the diffusion coefficient D for all species (Schuck, 2000). All size distributions were solved on a confidence level of p = 0.95 or 0.68, a best fitted f/f0, and a resolution N of 250 sedimentation coefficients between 0.1 and 25.0 S or molar mass between 1 and 1000 kDa, respectively.
Monomer-tetramer rapid self association model analysis
For rapidly associating systems, finite element solutions of the Lamm equation
 | (4) |
, 1998; Cox, 1969). For Lamm equation solutions with hydrodynamic repulsive nonideality, the local weight-average sedimentation coefficients were multiplied with a factor of 1/(1 + ksctot(r)), as described previously (Solovyova et al., 2001).
Dynamic light scattering
The dynamic light scattering (DLS) experiment was performed using a Proterion Protein Solution (Piscataway, NJ) DynaPro-MS/X dynamic light-scattering instrument. The laser power was set 20%, acquisition time was 10 s, and the temperature was 20°C. Intensity, contents, hydrodynamic radius (R), and polydispersity of the protein sample in PBS (pH 7.3) were real-time detected and analyzed by the software Dynamics V6. The total detecting time of each sample was 30–40 min.
Circular dichroism spectrometry
Circular dichroism (CD) spectrometry was performed using a Jasco J-810 spectropolarimeter (Easton, MD). Spectra of the sample dissolved in PBS (pH 7.3) were recorded at 30°C using 0.01-cm quartz cuvettes with a wavelength range of 190–250 nm at a step size of 0.2 nm. Signal averaging time was 1.0 s, and the slit bandwidth was 2 nm. The mean residue ellipticity ([
]) at each wavelength was calculated from Eq. 5,
 | (5) |
is the measured ellipticity in degree at wavelength , l is the cuvette pathlength (0.01 cm), and c is the protein concentration in g/ml. The deconvolution was performed using online CD analysis by DICHROWEB (Lobley and Wallace, 2001; Lobley et al., 2002) (http://www.cryst.bbk.ac.uk/cdweb/html/home.html). DICHROWEB offers several important pieces of analysis software for secondary structure determination, such as CONTINLL (Provencher and Glockner, 1981; Van Stokkum et al., 1990), SELCON3 (Sreerama and Woody, 1993; Sreerama et al., 1999), and K2D (Andrade et al., 1993).
ANS fluorescence spectrometry
Fluorescent dye binding experiments with 1-anilino-8-naphthalenesulfonic acid (ANS) as a probe were performed using Perkin-Elmer LS 50B luminescence spectrometer (Foster City, CA). The fluorescence emission of ANS is known to increase upon binding to accessible hydrophobic region of a protein (Semisotnov et al., 1991; Rogers et al., 1997). A fixed concentration of ANS (250 µM) was mixed with the protein (3 µM) in PBS (pH 7.3). The excitation wavelength was set at 395 nm and the emission spectrum was monitored from 400 to 600 nm wavelength. The area of the spectrum was calculated by the peak-fitting module of the Origin program (OriginLab, Northampton, MA). The average emission wavelength, 
, was calculated using Eq. 6,
 | (6) |
is the wavelength (Sánchez del Pino and Fersht, 1997). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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. The fitting results are shown as calculated c(s) versus sedimentation coefficient (Fig. 3, D and E) or calculated c(M) versus mass (Fig. 3 F). The distribution of each species was more clearly defined when a regularization of p = 0.68 was used (Fig. 3 E). ApoE4 and the other truncated fragments at different protein concentrations (0.15–1.00 mg/ml) were also analyzed (Table 1). Depending on the sequence and size of proteins, the partial specific volume of all the proteins was calculated (
0.73 ml/g) by program SEDNTERP (Laue et al., 1992) and so were molar extinction coefficients. Root mean square deviation (rmsd) of fitting to the continuous size distribution model was from 0.00384 to 0.00773 (Table 1). According to Schuck (2000) and Schuck et al. (2002), the fit in our studies was good and showed a high quality (rmsd < 0.01). Every sedimentation velocity experiment was repeated twice and similar results were obtained.
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; Table 1). The sw values of apoE and apoE1-271 proteins were from 6.1 to 6.6 S and did not show any significant difference. The C-terminal truncated apoE1-191 showed the lowest sw (2.6–3.0 S), whereas apoE31-231 had an sw of 3.8–4.2 S and apoE41-231 had an sw of 3.1–3.6 S. ApoE341-299 and apoE441-299 proteins showed lower sw values (4.4 and 3.7 S) at 0.15 mg/ml and higher sw values (4.8 and 4.3 S) at 0.50 or 1.00 mg/ml, respectively. Finally, the sw of the apoE472-299 protein had significantly increased (from 6.4 to 7.2) at a higher protein concentration, but apoE372-299 did not.
Size distribution of full-length apoE3 and apoE4 proteins
Size distribution of apoE3 and apoE4 full-length proteins showed very similar patterns (Fig. 4, A and B). Fitting to the continuous size distribution model showed overlapping peaks. To estimate each contributing component, the percentage and sw of the distribution curve by different integral range were analyzed with the integral function of SEDFIT (Table 2). There were four major species below s = 10. The large components (s > 10), with their lower content and broad distribution, were calculated as a single region. Both apoE3 and apoE4 proteins were separated into a five region distribution. The distribution of these components was very similar for both protein concentrations (0.15 mg/ml and 0.50 mg/ml). Concentration-dependent variation was observed. The aggregation species corresponding to s = 15.1 of apoE3 showed 6% increase and the species at s = 7.9 increased by 5%, whereas the smaller species at s = 4.0 and 5.9 showed 11% decreases. A similar trend was also observed in apoE4 species corresponding to s = 7.8, 9.8, and 14.9, which increased by 23% and those of s = 3.9 and 5.8, which decreased by 23%. Furthermore, at higher protein concentration, large species (s > 10) were more significantly increased for the apoE4 protein (by 15%), compared to apoE3 protein (6%). These observations support the greater aggregation of apoE4 protein.
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Fig. 4, E and F, shows the best-fit continuous c(s) distribution of apoE372-299 and apoE472-299. ApoE472-299 (Fig. 4 F) showed a more complicated distribution pattern than apoE372-299 (Fig. 4 F). The protein concentration had a significant influence on the distribution of species in apoE472-299 solution. The major species in apoE372-299 was s = 5.9 at 1.00 mg/ml protein concentration, whereas those in apoE472-299 were overlapping peaks separated into five species: s = 4.5, 6.1, 7.7, 9.3, and 11.0 (Fig. 4 F). After calculating the integration by SEDFIT, both apoE72-299 proteins showed four species at 0.15 mg/ml protein concentration (Table 2). However, when the protein concentration was increased to 1.00 mg/ml, 10 species were found in apoE472-299 protein, compared with five species of apoE372-299 (Fig. 4, E and F). This feature showed that aggregation of apoE472-299 protein was more sensitive to concentration. The smallest apoE472-299 species with s = 4.6 decreased by 4% at the higher protein concentration. At the same time, species s = 6.6 and 9.4 decreased by 24 and 8%, respectively, species s = 7.7 remained at 23%, and those with s > 10 increased by 14%. Similar to the full-length proteins, at higher protein concentrations, apoE472-299 had more abundant larger species (s > 10, 21%), compared with those of apoE372-299 (12%). ApoE472-299 therefore shows a greater tendency to aggregate.
Size distribution of C-terminal truncated apoE3 and apoE4 proteins
Fig. 5, A and C, shows the best-fit continuous c(s) distribution of the C-terminal truncated apoE proteins. At the initial protein concentration of 0.15 mg/ml, both apoE31-191 and apoE41-191 existed as two major peaks with s = 2.1 and s = 4.5 values (solid lines in Fig. 5, A and C). The two-species distribution was still maintained in the case of the apoE31-231 and apoE41-231 (dashed lines in Fig. 5, A and C), whereas the s values were shifted to 2.4 and 5.5 and the ratio of the two species also changed. From the results of c(M) distribution, the mass of the two species were estimated to be 22 kDa and 85 kDa for apoE1-191 and 30–35 kDa and 110–140 kDa for apoE1-231 (Fig. 5, B and D). These two species were assumed to be monomer and tetramer.
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Different from the 1–191 and 1–231 fragments in size distribution, apoE1-271 proteins showed overlapping peaks (dotted lines in Fig. 5, A and C). After calculating the integration area by SEDFIT, apoE31-271 could be resolved into six species with s = 3.6 (8%), 6.4 (64%), 8.9 (16%), 10.8 (8%), 13.2 (4%), and 17.4 (1%). The protein apoE41-271 showed a similar pattern with s = 3.5 (4%), 5.6 (29%), 7.0 (29%), 8.4 (17%), 10.2 (13%), 13.2 (4%), and 17.3 (4%), but it should be noted that there was a change in the relative amounts of each species. Similar to the c(S) distribution, continuous c(M) distribution (dotted lines in Fig. 5, B and D) also showed overlapping peaks in the range of 50–400 kDa. ApoE41-271 was made up of more and larger species (s > 10; 21%), compared to apoE31-271 (13%). However, they did not show a significant tendency to aggregate at higher protein concentration (data not shown).
Dynamic light scattering of apoE proteins
To verify the size distribution of the apoE3, apoE4, apoE372-299, and apoE472-299 proteins, DLS experiments were performed at an initial protein concentration of 1.0 mg/ml. Both apoE3 and E4 proteins showed a distribution with two major particle groups, one with a size of 3.9 nm and another in the range of 5.0–15.0 nm. However, apoE3 had less 5.0–15.0 nm group particles (35%), compared with those of apoE4 (46%). ApoE372-299 protein existed as one major particle group in the range of 4–10 nm, whereas apoE472-299 showed two major groups with a particle size of 5.1 nm (62%) and another in the range of 10.0–25.0 nm (36%).
Secondary structures of apoE proteins
Far-ultraviolet CD spectroscopy of apoE proteins is shown in Fig. 6. Comparing the two isoforms, the spectra of full-length apoE (circle lines in Fig. 6 A), 72–299 (square lines in Fig. 6 A), and 1–271 (hexagon lines in Fig. 6 B) fragments showed significant difference. All spectral data were further analyzed by the program CONTIN, SELCON3, and K2D. Because these programs showed similar simulations, only the results of CONTIN were shown in Table 3. ApoE3 protein indicated very high -helical content (91%), whereas that of apoE4 showed less (71%). The difference can also be found when the apoE31-271 (78%) and apoE41-271 (69%) proteins are compared. The N-terminal truncated fragments, apoE341-299 and apoE441-299 proteins maintained 54–55% -helix secondary structure and the percentage of random coil had increased by 13% compared with apoE4 and 38% compared with apoE3. ApoE372-299 protein showed relatively low percentage of -helical structure (38%), and high percentage of random coil (53%). In contrast, apoE472-299 protein still maintained 49% -helix and showed less random coil (47%).
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50–60% -helix and did not show an obvious difference between two isoforms.
ANS binding ability of apoE proteins
The binding of apoE proteins with the hydrophobic dye ANS was studied by fluorescence spectroscopy and the results are shown in Fig. 7. ANS alone displayed very low fluorescence in aqueous buffer (Fig. 7, dotted lines) and its fluorescence was significantly enhanced when bound to protein hydrophobic patches. Blue shift in the emission wavelength was observed after interaction with hydrophobic sites on proteins (Table 4). In both the apoE3 and apoE4 isoforms, the hydrophobic exposure of apoE41-299 proteins was always higher than that of full-length apoE proteins (Fig. 7). In addition, the hydrophobic exposure of the two isoforms did not show a significant difference in the case of the full-length or 41–299 fragments. Although apoE72-299 protein had lower hydrophobic exposure (compared with full-length and the 41–299 fragments), the apoE472-299 protein still showed more hydrophobic exposure than apoE372-299 protein (20% increase in fit area; Table 4).
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(triangle lines in Fig. 7 B; Table 4). Compared with apoE1-191 protein, the hydrophobic exposure of apoE1-231 protein showed a large increase (diamond lines in Fig. 7 B). ApoE1-271 showed the most hydrophobic exposure (hexagon lines in Fig. 7 B), and fit area had increased by 60%, compared with full-length apoE protein. |
DISCUSSION |
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This study elucidated structural variance between apoE3 and apoE4 isoforms at secondary, tertiary, and quaternary structure levels. ApoE4 showed a higher percentage of randomly coiled structure, compared with apoE3. This may result in a greater tendency to aggregate for apoE4 than apoE3. In the continuous c(s) distribution analyses, full-length apoE3 and apoE4 showed very similar patterns when the initial protein concentration was increased from 0.15 to 0.50 mg/ml. Concentration-dependent variation was observed and large species with s > 10 was more significantly increased for apoE4 protein (by 15%), compared with that of apoE3 protein (6%). The distribution patterns were different from those of a previous report (Perugini et al., 2000). However, a higher percent self-association of apoE4 isoform was found and this agrees with the previous report.
When helix 1 only or helices 1 and 2 of the N-terminal domain were truncated, the structural variance of apoE3 and apoE4 became more significant. The size distribution of apoE341-299 and apoE441-299 showed minor variance in the percentage of major species, whereas apoE372-299 and apoE472-299 showed significant differences in the secondary, tertiary, and quaternary structures. ApoE472-299 protein maintained more -helical structure. The accessibility of hydrophobic residues was also higher than for apoE372-299. These features showed that in addition to the salt bridge between Arg-61 and Glu-255, additional interactions may exist to maintain the structure of apoE472-299 protein. In addition, apoE472-299 showed a large amount of aggregation, especially at a high protein concentration. From the DLS experiments, the particle size of apoE472-299 protein was also larger than that of apoE372-299. It can be explained by the deletion of Arg-61...Glu-255 salt bridge (Dong et al., 1994; Dong and Weisgraber, 1996). Deletion of the N-terminal helices 1 and 2 of apoE3 protein resulted in the loss of most of the secondary structure and yet its major species at s = 5.9 was maintained. These observations suggested that the first two helices of N-terminal domain may play an important role in the structure stability of apoE3.
Farkas et al. (2004) have showed that the intracellular recycling of apoE and its N-terminal domain in vivo occur via multiple redundant pathways. In addition, Li et al. (2003) have suggested that the receptor 2 binding motif of apoE is localized at the N-terminus. These studies indicate that structural variance in the N-terminal truncated apoE3 and apoE4 may lead to different degradation or recycling pathways. The higher stability of truncated apoE4 proteins may extend the half-life inside or outside the cells, and may further lead to the formation of extracellular aggregated plaques or NFT, the hallmark of AD.
Another point of view was raised by Saito et al. (2001, 2003) and Morrow et al. (2002) and they suggested that the N-terminal domain of apoE4 (residues 1–191) form a molten globule and assume an open or a closed form in the absence or presence of lipid binding, respectively. Change in pH will induce a similar structural change. This phenomenon may be involved in the VLDL-binding efficiency of apoE4 proteins. Our results showed that apoE472-299 had a higher hydrophobic exposure, suggesting higher lipid-binding affinity, compared to apoE372-299.
The oligomerization of apoE protein is also closely related to the length of its C-terminal domain. From our results, the apoE1-191 protein existed mainly as monomer with some tetramer. When the helix 1 of C-terminal domain remained (192–231), the tetramer content was increased and monomer content was decreased. The apoE1-271 protein showed significant polymerization and the major species were assumed to be tetramers to hexamers (100–200 kDa, Fig. 5, B and D). Fan et al. (2004) indicated that the G* helix (268–289) is the key motif for the apoE aggregation. Our results suggest that the helices 1 and 2 of C-terminal domain (192–270) also induce polymerization of the apoE and that the Kd of apoE1-231 is significantly decreased and the polymerization of apoE1-271 is significant, as compared to apoE1-191 or apoE1-231. In contrast to the N-terminal truncation, C-terminal truncation of apoE3 and apoE4 proteins did not induce any significant variation. On the other hand, apoE C-terminal domain seemed to play an important role in the protein self-association of both apoE3 and apoE4.
Choy et al. (2004) suggested that there is an intermolecular coiled-coil formation in the apoE C-terminal domain. By sedimentation equilibrium studies, the dimer-tetramer-octamer model is suggested in the lipid-free environments, whereas the monomer-dimer-tetramer model is well maintained in the presence of 50% trifluoroethanol. Our sedimentation velocity studies showed that the only species present for the apoE C-terminal domain is a tetramer (s = 3.0) in PBS and the major species is a dimer (s = 1.5) in the presence of dihexanoylphosphatidylcholine (T. H. Lin, unpublished data).
The function of apoE to lower plasma cholesterol in vivo was checked by injection of proteins into apoE(–) mice (C. Y. Chou, G. G. Chang, and M. S. Shiao, unpublished data). The functional assay showed that full-length and truncated apoE proteins were active in vivo. Animal studies were conducted using apoE isoform proteins in pairs. Our preliminary results indicate that full-length apoE3 and apoE4, apoE341-299, apoE441-299, and apoE472-299 lowered the plasma cholesterol to a similar extent. However, the efficacy of apoE372-299 was significantly lower than that of full-length or apoE472-299 (30% less). This correlates well with helix 2 in the N-terminus being involved in the LDL receptor binding, especially for apoE3. One possibility is that stabilization by helix 2 to the LDL receptor binding of apoE3 proteins is more important than that of two-domain interactions. The more stabilized apoE4 isoform, which may assist in the domain-domain interaction, is less affected by helix 2 truncation. Other stabilization interactions may exist beyond the interaction of Arg-61 with Glu-255 and these may stabilize an extended helical structure in the carboxyl terminus to accommodate a larger, less-curved VLDL surface (Dong and Weisgraber, 1996).
Huang et al. (2001) have shown that C-terminal truncated fragments of apoE4 (272–299 amino acids removed) is generated inside cultured neurons and in brains of AD patients and interacted with p-tau and p-NF-H, resulting in large, filamentous intracellular inclusions resembling NFT in AD brains. When residues 245–271 in the C-terminus is deleted, the ability of apoE4 to form intracellular inclusions is completely lost. They have also found that N-terminal truncated apoE4 (1–85 residues removed) induces more NFT in culture neurons. The higher aggregation tendency of N-terminal truncated apoE4 and the importance of the C-terminus (232–271) in the self-association observed in our study may be due to the same reason. The findings for structural variation in N-terminal truncated apoE3 and apoE4 provide useful information to explain the higher levels of aggregation of the E4 isoform and this supports the involvement of E4 in AD. Our studies thus provide a direction for the rational design of drugs that may inhibit the aggregation of apoE4 and targeting this may delay the formation of NFT or extracellular amyloid plaques.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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This work was supported by the program for Promoting University Academic Excellence, National Science Council, and Taipei Veterans General Hospital, Taiwan, Republic of China.
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FOOTNOTES |
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-D-galactoside; LDL, low-density lipoprotein; NFT, neurofibrillary tangles; PBS, phosphate buffered saline; PCR, polymerase chain reaction; VLDL, very low-density lipoprotein. Submitted on May 28, 2004; accepted for publication September 27, 2004.
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REFERENCES |
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