Effects of polymorphism on the lipid interaction of human apolipoprotein E

ApoE exists as three common isoforms, apoE2, apoE3, and apoE4; apoE2 and apoE3 preferentially bind to high density lipoproteins, whereas apoE4 prefers very low density lipoproteins (VLDL). To understand the molecular basis for the different lipoprotein distributions of these isoforms in human plasma, we examined the lipid-binding properties of the apoE isoforms and some mutants using lipid emulsions. With both large (120 nm) and small (35 nm) emulsion particles, the binding affinity of apoE4 was much higher than that of apoE2 and apoE3, whereas the maximal binding capacities were similar among the three isoforms. The 22-kDa N-terminal fragment of apoE4 displayed a much higher binding capacity than did apoE2 and apoE3. The apoE4(E255A) mutant, which has no electrostatic interaction between Arg61 and Glu255, showed binding behavior similar to that of apoE3, indicating that N- and C-terminal domain interaction in apoE4 is responsible for its high affinity for lipid. In addition, the apoE3(P267A) mutant, which is postulated to contain a long alpha-helix in the C-terminal domain, had significantly decreased binding capacities for both sizes of emulsion particle, suggesting that the apoE4 preference for VLDL is not due to a stabilized long alpha-helical structure. Isothermal titration calorimetry measurements showed that there is no significant difference in thermodynamic parameters for emulsion binding among the apoE isoforms. However, fluorescence measurements of 8-anilino-1-naphthalenesulfonic acid binding to apoE indicated that apoE4 has more exposed hydrophobic surface compared with apoE3 mainly due to the different tertiary organization of the C-terminal domain. The less organized structure in the C-terminal domain of apoE4 leads to the higher affinity for lipid, contributing to its preferential association with VLDL. In fact, we found that apoE4 binds to VLDL with higher affinity compared with apoE3.     

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.     

Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4

Human apolipoprotein (apo) E4 binds preferentially to very low-density lipoproteins (VLDLs), whereas apoE3 binds preferentially to high-density lipoproteins (HDLs), resulting in different plasma cholesterol levels for the two isoforms. To understand the molecular basis for this effect, we engineered the isolated apoE N-terminal domain (residues 1-191) and C-terminal domain (residues 192-299) together with a series of variants containing deletions in the C-terminal domain and assessed their lipid and lipoprotein binding properties. Both isoforms can bind to a phospholipid (PL)-stabilized triolein emulsion, and residues 261-299 are primarily responsible for this activity. ApoE4 exhibits better lipid binding ability than apoE3 as a consequence of a rearrangement involving the segment spanning residues 261-272 in the C-terminal domain. The strong lipid binding ability of apoE4 coupled with the VLDL particle surface being ～60% PL-covered is the basis for its preference for binding VLDL rather than HDL. ApoE4 binds much more strongly than apoE3 to VLDL but less strongly than apoE3 to HDL(3), consistent with apoE-lipid interactions being relatively unimportant for binding to HDL. The preference of apoE3 for binding to HDL(3) arises because binding is mediated primarily by interaction of the N-terminal helix bundle domain with the resident apolipoproteins that cover ～80% of the HDL(3) particle surface. Thus, the selectivity in the binding of apoE3 and apoE4 to HDL(3) and VLDL is dependent upon two factors: (1) the stronger lipid binding ability of apoE4 relative to that of apoE3 and (2) the differences in the nature of the surfaces of VLDL and HDL(3) particles, with the former being largely covered with PL and the latter with protein.     

Lipid binding-induced conformational change in human apolipoprotein E. Evidence for two lipid-bound states on spherical particles

Apolipoprotein (apo) E contains two structural domains, a 22-kDa (amino acids 1-191) N-terminal domain and a 10-kDa (amino acids 223-299) C-terminal domain. To better understand apoE-lipid interactions on lipoprotein surfaces, we determined the thermodynamic parameters for binding of apoE4 and its 22- and 10-kDa fragments to triolein-egg phosphatidylcholine emulsions using a centrifugation assay and titration calorimetry. In both large (120 nm) and small (35 nm) emulsion particles, the binding affinities decreased in the order 10-kDa fragment approximately 34-kDa intact apoE4 > 22-kDa fragment, whereas the maximal binding capacity of intact apoE4 was much larger than those of the 22- and 10-kDa fragments. These results suggest that at maximal binding, the binding behavior of intact apoE4 is different from that of each fragment and that the N-terminal domain of intact apoE4 does not contact lipid. Isothermal titration calorimetry measurements showed that apoE binding to emulsions was an exothermic process. Binding to large particles is enthalpically driven, and binding to small particles is entropically driven. At a low surface concentration of protein, the binding enthalpy of intact apoE4 (-69 kcal/mol) was approximately equal to the sum of the enthalpies for the 22- and 10-kDa fragments, indicating that both the 22- and 10-kDa fragments interact with lipids. In a saturated condition, however, the binding enthalpy of intact apoE4 (-39 kcal/mol) was less exothermic and rather similar to that of each fragment, supporting the hypothesis that only the C-terminal domain of intact apoE4 binds to lipid. We conclude that the N-terminal four-helix bundle can adopt either open or closed conformations, depending upon the surface concentration of emulsion-bound apoE.     

Structural and functional variations in human apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 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.     

Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms.     

Contributions of the carboxyl-terminal helical segment to the self-association and lipoprotein preferences of human apolipoprotein E3 and E4 isoforms

To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.     

Human apolipoprotein E7:lysine mutations in the carboxy-terminal domain are directly responsible for preferential binding to very low density lipoproteins

Apolipoprotein E7 (apoE7) (apoE3 E244K/E245K) is a naturally occurring mutant in humans that is associated with increased plasma lipid levels and accelerated atherosclerosis. It is reported to display defective binding to low density lipoprotein (LDL) receptors, high affinity binding for heparin, and like apoE4, preferential association with very low density lipoproteins (VLDL). There are two potential explanations for the preference of apoE7 for VLDL: lysine mutations, which occur in the major lipid-binding region (residues 244-272) of the carboxy-terminal domain of apoE7, could either directly determine the lipoprotein-binding preference or could interact with negatively charged residues in the amino-terminal domain, resulting in a domain interaction similar to that in apoE4 (interaction of Arg-61 with Glu-255), which is responsible for the apoE4 VLDL preference. To distinguish between these possibilities, we determined the binding preferences of recombinant apoE7 and two amino-terminal domain mutants, apoE7 (E49Q/E50Q) and apoE7 (D65N/E66Q), to VLDL-like emulsion particles. ApoE7 and both mutants displayed a higher preference for the emulsion particles than did apoE3, indicating that the carboxy-terminal lysine mutations in apoE7 are directly responsible for its preference for VLDL. Supporting this conclusion, the carboxy-terminal domain 12-kDa fragment of apoE7 (residues 192;-299) displayed a higher preference for VLDL emulsions than did the wild-type fragment. In addition, lipid-free apoE7 had a higher affinity for heparin than did apoE. However, when apoE7 was complexed with dimyristoylphosphatidylcholine or VLDL emulsions, the affinity difference was eliminated. In contrast to previous studies, we found that apoE7 does not bind defectively to the LDL receptor, as determined in both cell culture and solid-phase assays.We conclude that the two additional lysine residues in the carboxy-terminal domain of apoE7 directly alter its lipid- and heparin-binding affinities. These characteristics of apoE7 could contribute to its association with increased plasma lipid levels and atherosclerosis.     

Characterization of the Binding of Amyloid-β Peptide to Cell Culture-Derived Native Apolipoprotein E2, E3, and E4 Isoforms and to Isoforms from Human Plasma

Abstract: The ε4 allele of apolipoprotein E (apoE, protein; APOE, gene) is a major risk factor for Alzheimer's disease (AD). Genetically, the frequency of the ε4 allele is enriched in early-onset sporadic, late-onset familial, and common late-onset sporadic AD. ApoE is found in the extracellular amyloid-β (Aβ) deposits that are characteristic features of AD. In this study, we examined the interaction between Aβ and apoE isoforms. The apoE isoforms used in this study were either produced by stably transfected Chinese hamster ovary cells (CHO) or were from human plasma. We report that when similar concentrations of the apoE isoforms were used, native nonpurified apoE3 from recombinant CHO-derived sources bound Aβ, but apoE4 did not. In fact, in our system, binding of recombinant apoE4 to Aβ was never detectable, even after incubation for 4 days. Furthermore, using the same assay conditions, native apoE2, like apoE3, binds Aβ avidly. Furthermore, when human plasma apoE isoforms are tested in Aβ binding experiments, apoE3 bound Aβ more avidly than apoE4, and a major apoE/Aβ complex (the 40-kDa form) was observed with plasma apoE3 but not apoE4. These data extend our understanding of apoE isoform-dependent binding of Aβ by associating apoE2 with efficient apoE/Aβ complex formation and demonstrate that native apoE3 (whether recombinant or derived from human plasma) forms sodium dodecyl sulfate-stable apoE/Aβ complexes more readily than native apoE4. The different Aβ-binding properties of native apoE4 versus native apoE3 provide insight into the molecular mechanisms by which the APOE ε4 allele exerts its risk factor effects in AD.     

Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

Novel role for apolipoprotein E in the central nervous system. Modulation of sulfatide content

It has long been postulated that apolipoprotein E (apoE) may play a role in lipid metabolism in the brain. However, direct evidence that apoE plays such a role is lacking. We investigated whether apoE isoforms influence lipid content in the brain. We compared the brains of wild-type mice to apoE knockout (-/-) and human apoE3 and apoE4 transgenic mice and compared cerebrospinal fluid (CSF) of humans with different apoE isoforms. We found that there was no effect of apoE on the content of multiple phospholipids, sphingolipids, and cholesterol. There was, however, a marked effect of apoE on the sulfatide (ST) content in both the brain and CSF. The sulfatide mass in hippocampus and cortex of apoE knockout mice was found to be 61 and 114 mol% higher than wild-type mice counterparts at 12 months of age. In contrast, the sulfatide content in brain tissues from human apoE4-expressing mice was approximately 60% less than those found in wild-type mice of the same age. The ST mass in human CSF was significantly dependent on the APOE genotypes of the subjects. Examination of potential sulfatide carrier(s) in human CSF demonstrated that sulfatides are specifically associated with apoE-containing high density lipoproteins, suggesting that sulfatide levels in the central nervous system (CNS) are likely to be directly modulated by the same metabolic pathways that regulate levels of apoE-containing CNS lipoproteins. This novel role for apoE in the CNS may provide new insights into the connection of apoE with Alzheimer's disease and poor recovery after brain injury.     

Engineering conformational destabilization into mouse apolipoprotein E. A model for a unique property of human apolipoprotein E4

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer and cardiovascular diseases. ApoE4 differs from the two other common isoforms (apoE2 and apoE3) by its lower resistance to denaturation and greater propensity to form partially folded intermediates. As a first step to determine the importance of stability differences in vivo, we reengineered a partially humanized variant of the amino-terminal domain of mouse apoE (T61R mouse apoE) to acquire a destabilized conformation like that of apoE4. For this process, we determined the crystal structure of wild-type mouse apoE, which, like apoE4, forms a four-helix bundle, and identified two structural differences in the turn between helices 2 and 3 and in the middle of helix 3 as potentially destabilizing sites. Introducing mutations G83T and N113G at these sites destabilized the mouse apoE conformation. The mutant mouse apoE more rapidly remodeled phospholipid than T61R mouse apoE, which supports the hypothesis that a destabilized conformation promotes apoE4 lipid binding.     

Effect of carboxyl-terminal truncation on structure and lipid interaction of human apolipoprotein E4

Apolipoprotein (apo) E4 has been identified as a major risk factor for Alzheimer's disease. Recently, apoE4 was found to undergo proteolytic cleavage in Alzheimer's disease brains, resulting in neurotoxic C-terminal-truncated fragments. In this study, we examined the effect of progressive truncation of the C-terminal domain in apoE4 on its lipid-free structure and lipid binding properties. Circular dichroism measurements demonstrated that deletion of residues 273-299 or 261-299 significantly decreased the number of helical residues, suggesting that the C-terminal residues 261-299 have alpha-helical structure. Although the progressive deletions in the C-terminal domain appear to somewhat increase thermal stability, apoE4 (delta273-299) and apoE4 (delta261-299) showed stability similar to that of the apoE4 22-kDa fragment (residues 1-191) when denatured with guanidine-HCl, indicating that residues 192-272 have a negligible effect on the stability of the C-terminal-truncated apoE4. Comparison of Trp-264 fluorescence in single Trp mutants of full-length and C-terminal-truncated apoE4 (delta273-299) indicated that the C-terminal domain structure in the latter is both less organized and cooperative. In addition, comparison of the binding of the C-terminal-truncated mutants to a hydrophobic fluorescent dye and to lipid emulsions revealed that residues 261-272 create a hydrophobic site which is critical for lipid binding. These results suggest that removal of a hydrophobic C-terminal alpha-helical segment (residues 273-299) to create C-terminal-truncated apoE4 forms found in brain leads to less organized C-terminal structure while still retaining a second alpha-helical lipid-binding region (residues 261-272) that is available for interaction with cell membranes and other proteins such as amyloid beta peptide.     

Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E

We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.     

Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid

Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease. We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods. Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P. (2000) Biophys. J. 78, 1606-1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4. In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers. Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate. We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mm) and to a lesser extent at submicellar concentrations (4 mm). The alpha-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine. These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.     

Lipid binding ability of human apolipoprotein E N-terminal domain isoforms: correlation with protein stability?

Human apolipoprotein (apo) E exists as one of three major isoforms, E2, E3 or E4. Individuals carrying the 4 allele have an increased risk of heart disease and premature onset of Alzheimer's disease. To investigate the molecular basis for this phenomenon, the N-terminal domain of apoE3, apoE2 and apoE4 were expressed in bacteria, isolated and employed in lipid binding and stability studies. Far UV circular dichroism spectroscopy in buffer at pH 7 revealed a similar amount of -helix secondary structure for the three isoforms. By contrast, differences were noted in apoE-NT isoform-specific transformation of bilayer vesicles of dimyristoylphosphatidylglycerol (DMPG) into discoidal complexes. ApoE4-NT induced transformation was most rapid, followed by apoE3-NT and apoE2-NT. To determine if differences in the rate of apoE-NT induced DMPG vesicle transformation is due to isoform-specific differences in helix bundle stability, guanidine HCl denaturation studies were conducted. The results revealed that apoE2-NT was the most stable, followed by apoE3-NT and apoE4-NT, establishing an inverse correlation between helix bundle stability and DMPG vesicle transformation rate at pH 7. When the zwitterionic dimyristoylphosphatidylcholine (DMPC) was employed as the model lipid surface, interaction of apoE-NT isoforms with the lipid substrate was slow. However, upon lowering the pH from 7 to 3, a dramatic increase in the rate of DMPC vesicle transformation rate was observed for each isoform. To evaluate if the increased DMPC vesicle transformation rates observed at low pH is due to pH-dependent alterations in helix bundle stability, guanidine HCl denaturation studies were performed. ApoE2-NT and apoE3-NT displayed increased resistance to denaturation as a function of decreasing pH, while apoE4-NT showed no change in stability. Studies with the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, indicated an increase in apoE hydrophobic surface exposure upon decreasing the pH to 3.0. Taken together, the data indicate that changes in the stability of secondary structure elements in apoE-NT isoforms are not responsible for pH-induced increases in lipid binding activity. It is likely that pH-induced disruption of inter-helical tertiary contacts may promote helix bundle conformational changes that present the hydrophobic interior of the protein to potential lipid surface binding sites.     

Characterization of apolipoprotein E genetic variations in Taiwanese: association with coronary heart disease and plasma lipid levels

Apolipoprotein E (apoE, protein; APOE, gene) is important in lipoprotein metabolism. Three isoforms, apoE2 (Cys112 Cys158), apoE3 (Cys112 Arg158), and apoE4 (Arg112 Arg158), are present in the general population. This report investigates the frequency distribution of apoE isoforms and the association of APOE genotypes with plasma lipid profile and coronary heart disease (CHD) in a population of Taiwan. ApoE isoforms were determined genetically by polymerase chain reaction and HhaI restriction enzyme digestion in control and coronary heart disease (CHD) patients. Plasma lipid and lipoprotein concentrations were also determined. The control group exhibited frequencies of 84.6% APOE3, 7.9% APOE4, 7.5% APOE2, 70.6% APOE3E3, 14.4% APOE3E4, 13.6% APOE2E3, and 1.4% APOE2E4. Comparable frequencies were observed in the CHD group. In both APOE2 carrier and APOE3E3 groups, the CHD patients expressed abnormal lipid profiles while the control group expressed normal lipid profiles. The APOE4 carriers, however, expressed abnormal lipid profiles in both normal control and CHD groups. Extremely high apoE levels in the hypertriglyceridemic group (TG > 400 mg/dL) seemed to be undesirable and were often observed in CHD patients.     

Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils

The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4>apoE3>apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly alpha-helical. The aggregates formed at substantially different rates (apoE4>apoE3>apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration.     

Dissociation of apolipoprotein E oligomers to monomer is required for high-affinity binding to phospholipid vesicles

The apolipoprotein apoE plays a key role in cholesterol and lipid metabolism. There are three isoforms of this protein, one of which, apoE4, is the major risk factor for Alzheimer's disease. At micromolar concentrations all lipid-free apoE isoforms exist primarily as monomers, dimers, and tetramers. However, the molecular weight form of apoE that binds to lipid has not been clearly defined. We have examined the role of self-association of apoE with respect to interactions with phospholipids. Measurements of the time dependence of turbidity clearance of small unilamellar vesicles of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) upon addition of apoE show that higher molecular weight oligomers bind poorly if at all. The kinetic data can be described by a reaction model in which tetramers and dimers of apoE must first dissociate to monomers which then bind to the liposome surface in a fast and reversible manner. A slow but not readily reversible conformational conversion of the monomer then occurs. Prior knowledge of the rate constants for the association-dissociation process allows us to determine the rate constant of the conformational conversion. This rate constant is isoform dependent and appears to correlate with the stability of the apoE isoforms with the rate of dissociation of the apoE oligomers to monomers being the rate-limiting process for lipidation. Differences in the lipidation kinetics between the apoE isoforms arise from their differences in the self-association behavior leading to the conclusion that self-association behavior may influence biological functions of apoE in an isoform-dependent manner.     

Distinct apolipoprotein E isoform preference for inhibition of smooth muscle cell migration and proliferation

The current study compared the effectiveness of the various human apolipoprotein E (apoE) isoforms in inhibiting platelet-derived growth factor- (PDGF-) stimulated smooth muscle cell proliferation and migration. The incubation of primary mouse aortic smooth muscle cells with apoE3 resulted in dose-dependent inhibition of smooth muscle cells stimulated by 10 ng/mL PDGF. Greater than 50% inhibition of smooth muscle cell proliferation was observed at 15 microg/mL of human apoE3. Human apoE2 was less effective, requiring a higher concentration to achieve inhibition comparable to that of apoE3. Human apoE4 was the least effective of the apoE isoforms with no significant inhibition of cell proliferation observed at concentrations up to 15 microg/mL. Interestingly, apoE inhibition of PDGF-directed smooth muscle cell migration did not show preference for any apoE isoforms. Human apoE2, apoE3, and apoE4 were equally effective in inhibiting smooth muscle cell migration toward PDGF. These results are consistent with previous data showing that apoE inhibition of smooth muscle cell proliferation is mediated through its binding to heparan sulfate proteoglycans, whereas its inhibition of cell migration is mediated via binding to the low-density lipoprotein receptor related protein. The low efficiency of apoE4 to inhibit smooth muscle cell proliferation also suggested another mechanism to explain the association between the apolipoprotein epsilon4 allele with increased risk of coronary artery disease.