Critical Review Apolipoprotein E Isoforms and Lipoprotein Metabolism
Michael C. Phillips
Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, 11-130 Translational Research Center, Philadelphia, PA, USA
Abstract Apolipoprotein (apo) E is a 299-residue protein which functions as a key regulator of plasma lipid levels. Human apoE exists as three common isoforms and the parent form, apoE3, operates optimally in promoting clearance of triglyceride (TG)rich lipoproteins and is associated with normal plasma lipid levels. This result occurs because apoE3 possesses both the requisite lipid-binding ability and affinity for the low density lipoprotein receptor (LDLR) to mediate appropriate lipolytic processing and endocytosis of TG-rich lipoprotein remnant particles. ApoE2 which differs from apoE3 by the single amino acid substitution Arg158Cys located near the LDLR recognition site exhibits impaired binding to the receptor and an inability to promote clearance of TG-rich lipoprotein remnant particles; this isoform is associated with Type-III hyperlipoproteinemia. ApoE4 which differs from apoE3 by the single amino acid substitution Cys112Arg is also associated with dyslipidemia although binding of this isoform to the LDLR is unaffected.
The amino acid substitution affects the organization and stability of both the N-terminal helix bundle domain and separately folded C-terminal domain so that apoE4 has enhanced lipid binding ability. As a consequence, apoE4 binds better than apoE3 to the surface of very low density lipoprotein (VLDL) particles and impairs their lipolytic processing in the circulation so that apoE4 is associated with a more pro-atherogenic lipoprotein-cholesterol distribution (higher VLDL-cholesterol/ high density lipoprotein-cholesterol ratio). This review summarizes current understanding of the structural differences between apoE2, apoE3, and apoE4, and the molecular mechanisms responsible for the alterations in lipoprotein metabolism resulting from this polymorphism of apoE. Detailed knowledge of how expression of structurally distinct apoE variants modifies lipoprotein metabolism provides a basis for developing C 2014 ways to manipulate the functionality of apoE in vivo. V IUBMB Life, 66(9):616–623, 2014
Keywords: apolipoprotein E; atherosclerosis; cholesterol; high density lipoprotein; lipoprotein metabolism; low density lipoprotein receptor; triglyceride; very low density lipoprotein
Introduction Apolipoprotein (apo) E is present on plasma lipoprotein particles in the circulation where it plays an important role in
Abbreviations: AAV, adeno-associated virus; Apo, apolipoprotein; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycan; LDLR, low density lipoprotein receptor; LPL, lipoprotein lipase; PL, phospholipids; TG, triglyceride; VLDL, very low density lipoprotein C 2014 International Union of Biochemistry and Molecular Biology V Volume 66, Number 9, September 2014, Pages 616–623 Address correspondence to: Michael Phillips, University of Pennsylvania, Philadelphia, Pennsylvania, USA. E-mail: [email protected] Received 11 August 2014; Accepted 9 September 2014 DOI 10.1002/iub.1314 Published online 9 October 2014 in Wiley Online Library (wileyonlinelibrary.com)
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cholesterol transport. This protein is a key regulator of plasma lipid levels, as exemplified by the situation in mice and humans lacking apoE where plasma cholesterol levels are markedly elevated and atherosclerosis is promoted (for a review, see (1)). The anti-atherogenic function of apoE arises in large part from its role in reducing plasma cholesterol levels by promoting clearance of triglyceride (TG)-rich lipoproteins from circulation (2,3). Human apoE exists as three common isoforms (apoE2, apoE3, and apoE4) and this polymorphism affects disease risk in carriers. The allele frequencies of e2, e3, and e4 in the human population are about 7, 78, and 14%, respectively (4). ApoE3 is considered to be the parent form and is associated with normal plasma cholesterol levels whereas the apoE2 and apoE4 isoforms have altered functionalities and are associated with the occurrence of hyperlipidemias. These abnormal plasma lipid levels and lipoprotein distributions are associated with increased risk of cardiovascular
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disease (1,2). This review summarizes current understanding of apoE structure-function relationships and of the molecular mechanisms responsible for the changes in the metabolism of TG-rich lipoproteins induced by the presence of the apoE2 and apoE4 isoforms.
Structure of Human ApoE3 Human apoE3 is a 299-residue molecule that contains multiple amphipathic a-helices which is a characteristic feature of exchangeable lipoproteins (5). The structure of apoE has been studied extensively over the past three decades (for reviews, see Refs. [6–8)). However, because of its flexible conformation and tendency to self-associate in aqueous solution, the highresolution structure of the entire apoE3 molecule was solved only recently (9). The NMR structure confirms earlier crystallographic studies in showing that the N-terminal region (residues 1–167) forms an anti-parallel four-helix bundle with the non-polar faces of the amphipathic helices facing the interior. This domain is separated by a hinge region from a C-terminal domain encompassing residues 206 to 299 that contains three a-helices which present a large exposed hydrophobic surface. These helices interact with those in the N-terminal helix bundle domain by formation of hydrogen-bonds and salt-bridges. Limited proteolysis studies (6) also have shown the existence of separately folded N- and C-terminal domains and this feature is indicated in Fig. 1. Protein engineering studies with apoE have demonstrated that the helix bundle domain contains a site recognized by the low density lipoprotein receptor (LDLR) and that the Cterminal domain is largely responsible for the lipid-binding ability of the protein (Fig. 1). The segment spanning residues 135 to 150 on helix four of the helix bundle domain is enriched in basic arginine (Arg) and lysine (Lys) residues that interact with acidic residues in the ligand-binding domain of members of the LDLR family (6). The Arg at position 172 is also critical for full LDLR binding activity (10). In addition to binding to acidic sites on the LDLR, the Arg and Lys residues in the apoE segment spanning residues 135 to 150 can also bind with high affinity to acidic sulfate groups on heparan sulfate proteoglycans (HSPG) (11). The specific positioning of the Arg and Lys residues in the amphipathic a-helix spanning residues 135 to 150 is important for maintaining optimal binding to the LDLR (12) but not for binding to HSPG (13). Studies of the lipid-binding and self-association properties of apoE3 variants containing C-terminal truncations indicate that the region spanning residues 260 to 299 is responsible for these properties of the apoE molecule (14–16). Lipid-free apoE in aqueous solution exists primarily as monomers, dimers, and tetramers and, consistent with the monomer being the form that can bind to lipid surfaces, the rate of dissociation of the oligomer to monomer is the rate-limiting step for lipidation (17). The exposed hydrophobic surface created by residues 260 to 270 in the C-terminal domain mediates the initial interaction of apoE with the surface of a phospholipid (PL)-contain-
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FIG 1
Structure of human apoE3. The 299-residue polypeptide chain is shown in linear form and the sites of the cysteine-arginine interchanges at positions 112 and 158 that distinguish apoE2 and apoE4 from apoE3 are marked. The protein is folded into two separate domains with N-terminal residues 1 to 191 containing an anti-parallel four-helix bundle and Cterminal residues 192 to 299 forming a separately folded domain that interacts with the helix bundle. The segment spanning residues 135 to 150 (horizontal red arrow) in the N-terminal domain contains a cluster of basic amino acids that form a binding site for the LDLR. The C-terminal segment spanning residues 260 to 299 (horizontal red arrow) contains amphipathic a-helices that initiate binding of the protein to lipid surfaces. See text for further details. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ing particle. After this first step involving insertion of the C-terminal amphipathic a-helices into the PL milieu at the particle surface, a second step ensues in which the N-terminal helix bundle opens to convert hydrophobic helix-helix interactions to helix-lipid interactions (18–20). In this step, helices one and two and helices three and four in the helix bundle preferentially remain paired (21). Furthermore, the open helix bundle adopts a partially extended conformation (22) and reorients with respect to the position of the C-terminal domain (23). Lipid association induces additional a-helix formation in apoE and when present in discoidal particles containing a segment of PL bilayer, the a-helices in both the N- and C-terminal domains are aligned perpendicular to the PL fatty acyl chains (24). Internal reflection infrared spectroscopy indicates that apoE interacts either with the edge of the PL bilayer or with the PL polar groups (25). The latter interaction occurs in quasi-spherical apoE/dipalmitoyl phosphatidylcholine particles where the two apoE molecules adopt a horseshoe shape (7,26). The conformation of lipid-bound apoE is dependent upon the shape and size of the particle with which it is associated. In the case of apoE bound to spherical lipid or lipoprotein particles, the N-terminal helix bundle can adopt either open or closed conformations (27,28). At low surface coverage, the four-helix bundle is open and all helices are in contact with lipid surface. In contrast, at high apoE surface concentrations, displacement of the N-terminal domain from the lipid surface by the more hydrophobic C-terminal domain causes the fourhelix bundle to adopt a closed conformation. This change in conformation alters the ability of apoE to bind to the LDLR because the helix bundle has to be open for recognition by the
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FIG 2
The role of apoE in the metabolism of plasma lipoproteins. Chylomicron particles secreted from the intestine and VLDL particles secreted from the liver are lipolyzed in the circulation by LPL to form remnant particles. ApoE (red) on such particles mediates their uptake in the liver by the LDLR or the LDLRrelated protein (LRP) and heparan sulfate proteoglycan (HSPG) pathways. The LDL particles formed by complete lipolysis of VLDL lack apoE and the apoB100 on such particles is the ligand for the LDLR. Adapted from Ref. 33. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
apo-B100 to the LDLR. ApoE is also important in the production of VLDL particles because its intracellular expression in hepatocytes promotes VLDL assembly and secretion (34). Optimal expression of apoE3 is crucial for normal metabolism of TG-rich lipoproteins. Over-expression and/or accumulation of apoE stimulates VLDL TG production which leads to hypertriglyceridemia. In addition, the presence of excess of apoE on VLDL particles can impair their lipolysis which elevates plasma TG levels (35). The lipid cargo delivered to hepatocytes by receptormediated endocytosis of TG-rich lipoprotein remnant particles is catabolized in lysosomes whereas the majority of the apoE remains in peripheral recycling endosomes. This pool of apoE is recycled back to the plasma membrane, resecreted and becomes part of an apoE-containing HDL pool (36). ApoE secreted by macrophages also contributes to the pool of apoEHDL. The expression of apoE in macrophages plays an important anti-atherogenic role by promoting cholesterol efflux from cells in the arterial wall (37).
ApoE Polymorphism receptor (6). Opening of the helix bundle alters the microenvironment in the receptor binding region around residues 135 to 150 so that there is greater positive electrostatic potential which enhances binding to acidic residues in the LDLR (29). In addition, lipid interaction also elongates the apoE helix in which the basic residues are situated, thereby enhancing the ability to bind to the LDLR (30). The open and closed conformations of the apoE helix bundle apparently coexist on the surface of hypertriglyceridemic very-low-density-lipoprotein (VLDL) particles because some of the apoE is recognized by the LDLR and some is not (31). Only the receptor-inactive conformation (helix bundle closed) of apoE is present at the surface of the VLDL particles isolated from normolipidemic individuals.
The polymorphism of human apoE is summarized in Table 1. The three major isoforms, apoE2, apoE3, and apoE4 differ by amino acid substitutions at positions 112 and 158. ApoE3 contains cysteine (Cys) at position 112 and Arg at position at 158, whereas apoE2 and apoE4 contains Cys and Arg, respectively, at both sites (6). ApoE3 is the most common isoform and is associated with normal lipoprotein metabolism whereas the less common apoE2 and apoE3 isoforms are associated with abnormal metabolism. ApoE also regulates lipid transport and cholesterol homeostasis in the brain and the apoE4 isoform is associated with Alzheimer’s disease (2,8). ApoE3 and apoE4 bind to the LDLR with similarly high affinity but the binding of apoE2 is some two orders of magnitude weaker (38).
Metabolic Functions of ApoE3
ApoE2 and Type III Hyperlipoproteinemia
ApoE in the peripheral circulation originates mostly from expression in hepatocytes, although there is a small contribution from expression in macrophages. Plasma apoE circulates associated with chylomicron, VLDL, and HDL particles where it plays a key role in their metabolism. As summarized in Fig. 2, chylomicrons derived from the intestine and VLDL derived from the liver are lipolyzed in the circulation by lipoprotein lipase (LPL) (32,33). ApoE on the remnant lipoprotein particles binds to the LDLR, LDLR-related protein and HSPG present on the surface of hepatocytes where the remnant particles are endocytosed and removed from the circulation. Some VLDL remnants are cleared rapidly whereas others undergo further lipolysis and are converted progressively to intermediate density lipoprotein (IDL) and finally to LDL. The LDL particles do not contain apoE and their clearance is mediated by binding of
The impaired binding of apoE2 to the LDLR is the reason for its association with Type III hyperlipoproteinemia. The degree to which receptor binding is reduced depends upon the nature of the lipoprotein particle to which apoE2 is attached and a mechanistic explanation for the defective binding has come from X-ray crystallographic comparisons of apoE2 and apoE3 (39). ApoE2 differs from apoE3 by the Arg to Cys interchange at position 158 which is close to the LDLR binding region (Fig. 1). This amino acid substitution in the amphipathic a-helix present in this region eliminates a salt-bridge between Arg158 and aspartic acid (Asp)154 which is present in apoE3 and leads to formation of a different salt-bridge between Asp158 and Arg150 in apoE2 (39). This new salt-bridge alters the conformation of Arg150 with respect to the other basic residues in the receptor-binding region so that it is no longer available to
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TABLE 1
Human apoE isoformsa
Protein
Apo E-2
Apo E-3
Apo E-4
Residue at position 112
Cysteine
Cysteine
Arginine
Residue at position 158
Cysteine
Arginine
Arginine
% LDL Receptor binding affinity
1
100
100
Population allelic frequency (%)
7
78
14
Type III Hyperlipoproteinemia
Normal
CVD and Alzheimer’s Disease
Clinical association a
Information taken from citations in the text.
interact with the LDLR, thereby reducing the ability of apoE2 to bind to the receptor. The impaired ability of apoE2 to bind to the LDLR leads to poor clearance of TG-rich lipoprotein remnants from the plasma compartment and predisposes carriers of this isoform to Type III hyperlipoproteinemia (33). The presence of apoE2 is essential but not sufficient to induce Type III hyperlipoproteinemia because, while this isoform does not bind to the LDLR well, its recognition by the LDLR-related receptor and HSPG is largely unaffected so that remnant lipoprotein clearance can still occur to some extent. Overt hyperlipidemia requires homozygosity for apoE2 combined with additional genetic or environmental factors such as obesity and a high fat diet to stress the system. Other mutations in or near the LDLR binding site of the apoE molecule which reduce the net positive electrostatic potential also give rise to Type III hyperlipoproteinemia (33,40). The severity of the hyperlipidemia associated with these rare apoE mutations is reflected in parallel reductions in the abilities of the isoforms to bind to cells and to heparin (41). Indeed, apoE variants that are defective in binding to all three types of cell surface receptor (LDLR, LDLR-related protein, HSPG) are associated with dominant inheritance of the trait and heterozygosity is sufficient for overt Type III hyperlipoproteinemia (33).
Comparison of the Structures and Properties of ApoE3 and ApoE4 While it is clear that the association of apoE2 with hyperlipidemia is a consequence of defective binding to the LDLR, this explanation does not account for the hypercholesterolemia associated with apoE4 (4) because this isoform binds like apoE3 to the receptor (38). The reason for the more proatherogenic lipoprotein-cholesterol distribution in the plasma of apoE4 carriers is the different lipoprotein distributions of apoE3 and apoE4 in plasma. When added to plasma, apoE3 binds preferentially to HDL and apoE4 binds more to VLDL
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(42,43). As summarized below, the molecular basis for this difference has been shown to be due to structural differences between the two apoE isoforms. The Cys112Arg substitution which converts apoE3 into apoE4 is located in the N-terminal helix bundle domain (Fig. 1) and replacement of the small Cys side chain with the bulky and positively charged Arg side chain destabilizes the helix bundle (44,45). The presence of Arg112 in apoE4 leads to rearrangement of the Arg61 side chain in the helix bundle, so that, unlike the situation apoE3, it forms a salt-bridge with glutamic acid (Glu) 255 in the C-terminal domain (46,47). This binding and probably other allosteric effects (48) lead to altered interactions between the N- and C-terminal domains in the apoE4 molecule. As a consequence, the C-terminal domain is organized differently in the two isoforms. For example, in apoE4 position 264 is less organized and more exposed to the aqueous environment (15) and the spacing between the N- and C-terminal domains is altered (49,50). Additionally, as assessed by urea denaturation, the apoE4 C-terminal domain is less stable and unfolds more readily than its counterpart in apoE3 (51). Overall, it is clear that the single amino acid substitution Cys112Arg significantly alters the structure of the protein so that the functions of apoE3 and apoE4 are different. Of particular note, the lipid binding properties of apoE3 and apoE4 are different with the latter isoform binding with higher affinity to lipid emulsion particles (52). Like apoE3, apoE4 binds to lipid particles by a two-step mechanism with the binding being initiated by the C-terminal domain (20). ApoE4 exhibits better lipid binding ability than apoE3 as a consequence, in part, of a rearrangement involving the segment spanning residues 260 to 272 in the C-terminal domain (Fig. 1) (16). The destabilization of the N-terminal helix bundle domain in apoE4 which allows it to unfold relatively readily compared to the situation in apoE3 also contributes importantly to the enhanced lipid binding ability of apoE4 (51). The strong lipid binding ability of apoE4 is the basis for the preferential binding of this isoform to VLDL particles, the surfaces of which are 60% PL-covered. ApoE4 binds much better than apoE3 to VLDL but somewhat less well than apoE3 to HDL.
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FIG 3
Contributions of different regions of the apoE3 and apoE4 molecules to their abilities to clear plasma cholesterol. The linear representations of the amino acid sequences show the position in the N-terminal helix bundle domain of the C112R (Cys112Arg) substitution that distinguishes the two isoforms. The fractional contribution to plasma cholesterollowering (F) is indicated beneath the indicated segments of the apoE3 and apoE4 molecules. The F values are calculated from the relative efficiencies of plasma cholesterol reduction for the apoE3 and apoE4 C-terminal truncation variants (1–272), (1–260), and (1–191) (57). For instance, the F value (0.8) of the segment spanning residues 261 to 272 in apoE3 is obtained by subtracting the cholesterol-lowering efficiency of apoE3 (1–260) (0.2) from that of apoE3 (1 to 272) (1.0). The importance of the segment spanning residues 261 to 272 is highlighted by the red bar marking this section of the apoE molecule. See text for further details. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Binding of apoE to HDL is mediated primarily by interaction of the N-terminal helix bundle domain with the resident apolipoproteins that cover 80% of the HDL particle surface. Thus, the selectivity in the binding of apoE3 and apoE4 to HDL and VLDL particles 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 particles, with the former being largely covered with PL and the latter with protein (16).
Comparison of the Influences of ApoE3 and ApoE4 on Lipoprotein Metabolism In Vivo The question of how the differences in structure and lipidbinding properties between apoE3 and apoE4 affect lipoprotein metabolism in vivo has proven difficult to answer. It is established that in mice expressing either human apoE3 or apoE4 in place of the endogenous mouse apoE, plasma total cholesterol levels are the same but expression of apoE4 is associated with higher VLDL-cholesterol and incidence of atherosclerosis (53,54). The strong anti-atherogenic function of apoE3 has been demonstrated by liver-specific expression
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using adeno-associated viral (AAV) vectors in apoE-deficient mice in which plasma cholesterol levels are reduced to normal wild-type mouse levels and atherosclerosis is prevented after 1 year of sustained expression (55). The clearance of apoEcontaining lipoproteins in mice occurs mainly by interaction with the LDLR (cf. Fig. 2) (56). With this background, we used the apoE-null mouse model and AAV to determine the effects on plasma cholesterol levels and lipoprotein-cholesterol distributions of expressing different doses of apoE3 and apoE4 and C-terminal deletion variants of both isoforms in the liver (57). In agreement with the findings in transgenic mice, apoE3, and apoE4 reduce plasma cholesterol levels to the same extent. As expected, the ability of apoE to bind to lipoprotein particles is critical for this effect because removal of the lipid-binding C-terminal domain greatly reduces effectiveness. Thus, expression of the isolated Nterminal domain (residues 1–191) of apoE3 gives zero cholesterol-lowering and about 30% activity in the case of apoE4 (Fig. 3). The fractional contributions to plasma cholesterol-lowering (F) of different regions of the apoE3 and apoE4 molecules are summarized in Fig. 3. It is apparent that residues 261 to 272 make the largest contribution to the reduction in plasma total cholesterol levels in both isoforms. This effect is consistent with the ability of apoE to bind to lipoprotein surfaces being critical for cholesterol-lowering because, as discussed earlier, this segment forms a hydrophobic surface patch which is key for lipid emulsion and VLDL binding activity. The different F value for residues 261 to 272 in apoE3 and apoE4 is consistent with the altered structural organization of this segment in the two proteins. The reduced F for this segment in apoE4 is compensated for by an increased contribution from its relatively unstable helix bundle domain (Fig. 3). Given that the overall plasma cholesterol-lowering ability of apoE4 is the same as that of apoE3, the question remains as to why apoE4 is associated with increased risk of cardiovascular disease. Insight into this issue was provided by an examination of the lipoprotein-cholesterol distributions in apoE3and apoE4-expressing mice (57). Importantly, under conditions where the plasma total cholesterol levels are the same apoE4 is associated with a more pro-atherogenic lipoprotein-cholesterol distribution (i.e. a higher VLDL-cholesterol/HDL-cholesterol ratio). This effect arises because VLDL processing via the LPL-mediated lipolysis cascade mentioned in Fig. 2 is relatively restricted in apoE4-expressing animals. The relative distribution of apoE between VLDL and HDL particles plays a crucial role in this process. The basis for the deranged behavior of apoE4 in the catabolism of VLDL is presented in detail in Fig. 4. In essence, the apoE content of VLDL has to be sufficient to support LDLR binding (and the apoE molecules have to be in the receptor-active conformation with the N-terminal helix bundle open) but not too much to displace apoC-II, the cofactor for LPL, and thereby inhibit lipolysis (33,58,59). The distribution of apoE3 between VLDL and HDL is appropriate for optimal progress down the lipolysis cascade. In contrast, the
Apolipoprotein E Isoforms and Lipoprotein Metabolism
FIG 4
Schematic comparing the influence of apoE3 and apoE4 on the lipolysis cascade involved in the catabolism of VLDL particles (57). After secretion from the liver into the plasma compartment, the triglyceride (TG) in VLDL is hydrolyzed by lipoprotein lipase (LPL) with apoC-II as a cofactor leading to the creation of intermediate density lipoprotein (IDL) and progressively smaller remnant particles. ApoE bound to these particles mediates their interaction with the LDLR and clearance from plasma (the lower red curved arrow shows the decrease in VLDL and remnant cholesterol levels). As the VLDL/IDL remnants shrink due to the removal of core TG, excess surface components (PL, cholesterol and apoE) are released into the HDL pool (upper blue curved arrow shows the increase in HDL cholesterol level). ApoE3 partitions between the VLDL and HDL pools so that lipolysis, clearance of VLDL remnant cholesterol and HDL formation is optimal. In the diagram, points c and d represent the VLDL/IDL-cholesterol and HDLcholesterol levels, respectively, when apoE3 is expressed. Relative to apoE3, apoE4 binds more to VLDL because of its higher lipid affinity leading to inhibition of lipolysis (probably because of displacement of apoC-II) so that, at the same apoE expression level, progression down the lipolysis cascade is relatively limited in the case of apoE4. The ratio a/b represents the apoE4 VLDL-C/HDL-C ratio which is higher than the apoE3 ratio c/d. See text for further details. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
greater lipid-binding ability of apoE4 increases the concentration of apoE4 on the VLDL surface so that lipolysis is impaired, resulting in reduced VLDL remnant clearance and an elevated VLDL-cholesterol/HDL-cholesterol ratio. The critical role for the ability of apoE to partition appropriately between VLDL and HDL during the lipolytic cascade is supported by the finding that deleting residues 273 to 299 to reduce the VLDL-binding affinity of apoE4 normalizes the VLDL-cholesterol/HDL-cholesterol ratio to that of apoE3 (57). This result suggests that pharmacologic intervention in apoE4 subjects to reduce the apoE content of VLDL could be beneficial therapeutically.
Summary and Conclusions The ability of apoE to bind to the surfaces of chylomicron, VLDL, and HDL particles as well as to cell surface molecules
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such as the LDLR and HSPG underlies its function as a key regulator of plasma cholesterol and TG levels. The dysfuctionality of apoE2 in lipoprotein metabolism arises from impaired binding to the LDLR whereas the dysfunctionality of apoE4 arises from enhanced binding to VLDL particles. The structural basis for these altered binding properties of these two isoforms is understood. The presence of Cys158 disrupts the nearby LDLR recognition site in the apoE2 molecule while the presence of Arg112 in apoE4 destabilizes the N- and Cterminal domains conferring enhanced lipid binding ability. Human apoE3 and apoE4 are equally effective at reducing total plasma cholesterol in apoE-null mice but the apoE4expressing animals have a pro-atherogenic lipoprotein profile (higher VLDL-cholesterol/HDL-cholesterol ratio) compared to their apoE3-expressing counterparts. Moderate reduction of the VLDL binding affinity of apoE4 reduces this proatherogenic lipoprotein profile.
Acknowledgements The author is indebted to all colleagues for their valuable contributions to the studies from this laboratory described here.
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[31] Bradley, W. A., Hwang, S. L., Karlin, J. B., Lin, A. H., Prasad, S. C., et al. (1984) Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins. J. Biol. Chem. 259, 14728–14735. [32] Mahley, R. W. and Ji, S. Z. (1999) Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E. J. Lipid. Res. 40, 1–16.
[34] Sundaram, M. and Yao, Z. (2012) Intrahepatic role of exchangeable apolipoproteins in lipoprotein assembly and secretion. Arterioscler. Thromb. Vasc. Biol. 32, 1073–1078. [35] Huang, Y., Liu, X. Q., Rall, S. C., Jr., Taylor, J. M., von Eckardstein, A., et al. (1998) Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia. J. Biol. Chem. 273, 26388–26393. [36] Heeren, J., Beisiegel, U., and Grewal, T. (2006) Apolipoprotein E recycling. Implications for dyslipidemia and atherosclerosis. Arterioscler.Thromb.Vasc.Biol. 26, 442–448. [37] Bellosta, S., Mahley, R. W., Sanan, D. A., Murata, J., Newland, D. L., et al. (1995) Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice. J. Clin. Invest. 96, 2170–2179. [38] Weisgraber, K. H., Innerarity, T. L., and Mahley, R. W. (1982) Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteinearginine interchange at a single site. J. Biol. Chem. 257, 2518–2521. [39] Dong, L. M., Parkin, S., Trakhanov, S. D., Rupp, B., Simmons, T., et al. (1996) Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia. Nat. Struct. Biol. 3, 718–722. [40] Dong, L. M., Innerarity, T. L., Arnold, K. S., Newhouse, Y. M., and Weisgraber, K. H. (1998) The carboxyl terminus in apolipoprotein E2 and the seven amino acid repeat in apolipoprotein E-Leiden: role in receptorbinding activity. J. Lipid Res. 39, 1173–1180. [41] Mann, W. A., Meyer, N., Weber, W., Meyer, S., Greten, H., et al. (1995) Apolipoprotein E isoforms and rare mutations: parallel reduction in binding to cells and to heparin reflects severity of associated type III hyperlipoproteinemia. J. Lipid Res. 36, 517–525. [42] Steinmetz, A., Jakobs, C., Motzny, S., and Kaffarnik, H. (1989) Differential distribution of apolipoprotein E isoforms in human plasma lipoproteins. Arteriosclerosis 9, 405–411. [43] Weisgraber, K. H. (1990) Apolipoprotein E distribution among human plasma lipoproteins: role of the cysteine-arginine interchange at residue 112. J. Lipid Res. 31, 1503–1511. [44] Morrow, J. A., Segall, M. L., Lund-Katz, S., Phillips, M. C., Knapp, M., et al. (2000) Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain. Biochemistry 39, 11657–11666. [45] Morrow, J. A., Hatters, D. M., Lu, B., Hocht, P., Oberg, K. A., et al. (2002) Apolipoprotein E4 forms a molten globule. J. Biol. Chem. 277, 50380–50385. [46] Dong, L. M. and Weisgraber, K. H. (1996) Human apolipoprotein E4 domain interaction. Arginine 61 and glutamic acid 255 interact to direct the preference for very low density lipoproteins. J Biol Chem 271, 19053–19057. [47] Dong, L. M., Wilson, C., Wardell, M. R., Simmons, T., Mahley, R. W., et al. (1994) Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms. J. Biol. Chem. 269, 22358– 22365. [48] Frieden, C. and Garai, K. (2013) Concerning the structure of apoE. Protein Sci. 22, 1820–1825. [49] Hatters, D. M., Budamagunta, M. S., Voss, J. C., and Weisgraber, K. H. (2005) Modulation of apolipoprotein E structure by domain interaction. Differences in lipid-bound and lipid-free forms. J. Biol. Chem. 280, 34288–34295. [50] Drury, J. and Narayanaswami, V. (2005) Examination of lipid-bound conformation of apolipoprotein E4 by pyrene excimer fluorescence. J. Biol. Chem. 280, 14605–14610. [51] Nguyen, D., Dhanasekaran, P., Nickel, M., Mizuguchi, C., Watanabe, M., et al. (2014) Influence of domain stability on the properties of human apolipoprotein E3 and E4 and mouse apolipoprotein E. Biochemistry 53, 4025–4033. [52] Saito, H., Dhanasekaran, P., Baldwin, F., Weisgraber, K. H., Phillips, M. C., et al. (2003) Effects of polymorphism on the lipid interaction of human apolipoprotein E. J. Biol. Chem. 278, 40723–40729. [53] Knouff, C., Hinsdale, M. E., Mezdour, H., Altenburg, M. K., Watanabe, M., et al. (1999) Apo E structure determines VLDL clearance and atherosclerosis risk in mice. J. Clin.Invest. 103, 1579–1586.
[33] Mahley, R. W., Huang, Y., and Rall, S. C. (1999) Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia): questions, quandaries, and paradoxes. J. Lipid Res. 40, 1933–1949.
[54] Pendse, A. A., Arbones-Mainar, J. M., Johnson, L. A., Altenburg, M., and Maeda, N. (2009) ApoE knock-out and knock-in mice: Atherosclerosis, metabolic syndrome, and beyond. J. Lipid Res. 50, S178–S182.
[14] Westerlund, J. A. and Weisgraber, K. H. (1993) Discrete carboxyl-terminal segments of apolipoprotein E mediate lipoprotein association and protein oligomerization. J. Biol. Chem. 268, 15745–15750. [15] Sakamoto, T., Tanaka, M., Vedhachalam, C., Nickel, M., Nguyen, D., et al. (2008) Contributions of the carboxyl-terminal helical segment to the selfassociation and lipoprotein preferences of human apolipoprotein E3 and E4 isoforms. Biochemistry 47, 2968–2977. [16] Nguyen, D., Dhanasekaran, P., Nickel, M., Nakatani, R., Saito, H., et al. (2010) Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4. Biochemistry 49, 10881– 10889. [17] Garai, K., Baban, B., and Frieden, C. (2011) Dissociation of apolipoprotein E oligomers to monomer is required for high-affinity binding to phospholipid vesicles. Biochemistry 50, 2550–2558. [18] Saito, H., Dhanasekaran, P., Nguyen, D., Holvoet, P., Lund-Katz, S., et al. (2003) Domain structure and lipid interaction in human apolipoproteins A-I and E: a general model. J. Biol. Chem. 278, 23227–23232. [19] Saito, H., Lund-Katz, S., and Phillips, M. C. (2004) Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins. Prog. Lipid Res. 43, 350–380. [20] Nguyen, D., Dhanasekaran, P., Phillips, M. C., and Lund-Katz, S. (2009) Molecular mechanism of apolipoprotein E binding to lipoprotein particles. Biochemistry 48, 3025–3032. [21] Lu, B., Morrow, J. A., and Weisgraber, K. H. (2000) Conformational reorganization of the four-helix bundle of human apolipoprotein E in binding to phospholipid. J. Biol. Chem. 275, 20775–20781. [22] Fisher, C. A., Narayanaswami, V., and Ryan, R. O. (2000) The lipidassociated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E. J. Biol. Chem. 275, 33601–33606. [23] Narayanaswami, V., Szeto, S. S., and Ryan, R. O. (2001) Lipid associationinduced N- and C-terminal domain reorganization in human apolipoprotein E3. J. Biol. Chem. 276, 37853–37860. [24] Narayanaswami, V., Maiorano, J. N., Dhanasekaran, P., Ryan, R. O., Phillips, M. C., et al. (2004) Helix orientation of the functional domains in apolipoprotein E in discoidal high density lipoprotein particles. J. Biol. Chem. 279, 14273–14279. [25] Schneeweis, L. A., Koppaka, V., Lund-Katz, S., Phillips, M. C., and Axelsen, P. H. (2005) Structural analysis of lipoprotein E particles. Biochemistry 44, 12525–12534. [26] Peters-Libeu, C. A., Newhouse, Y., Hall, S. C., Witkowska, H. E., and Weisgraber, K. H. (2007) Apolipoprotein E dipalmitoylphosphatidylcholine particles are ellipsoidal in solution. J.Lipid Res. 48, 1035–1044. [27] Narayanaswami, V. and Ryan, R. O. (2000) Molecular basis of exchangeable apolipoprotein function. Biochim. Biophys. Acta 1483, 15–36. [28] Saito, H., Dhanasekaran, P., Baldwin, F., Weisgraber, K., Lund-Katz, S., et al. (2001) Lipid binding-induced conformational change in human apolipoprotein E. J. Biol. Chem. 276, 40949–40954. [29] Lund-Katz, S., Zaiou, M., Wehrli, S., Dhanasekaran, P., Baldwin, F., et al. (2000) Effects of lipid interaction on the lysine microenvironments in apolipoprotein E. J. Biol. Chem. 275, 34459–34464. [30] Gupta, V., Narayanaswami, V., Budamagunta, M. S., Yamamato, T., Voss, J. C., et al. (2006) Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability. J. Biol. Chem. 281, 39294–39299.
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[55] Kitajima, K., Marchadier, D. H., Miller, G. C., Gao, G. P., Wilson, J. M., et al. (2006) Complete prevention of atherosclerosis in apoE-deficient mice by hepatic human apoE gene transfer with adeno-associated virus serotypes 7 and 8. Arterioscler. Thromb. Vasc. Biol. 26, 1852–1857. [56] Zannis, V. I., Chroni, A., Kypreos, K. E., Kan, H. Y., Cesar, T. B., et al. (2004) Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer. Curr. Opin. Lipidol. 15, 151– 166. [57] Li, H., Dhanasekaran, P., Alexander, E. T., Rader, D. J., Phillips, M. C., et al. (2013) Molecular mechanisms responsible for the differential effects of
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apoE3 and apoE4 on plasma lipoprotein-cholesterol levels. Arterioscler. Thromb. Vasc. Biol. 33, 687–693. [58] Weisgraber, K. H., Mahley, R. W., Kowal, R. C., Herz, J., Goldstein, J. L., et al. (1990) Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein. J. Biol. Chem. 265, 22453–22459. [59] Rensen, P. C. N. and Van Berkel, T. J. C. (1996) Apolipoprotein E effectively inhibits lipoprotein lipase-mediated lipolysis of chylomicron-like triglyceriderich lipid emulsions in vitro and in vivo. J. Biol. Chem. 271, 14791–14799.
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Michael C. Phillips
Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, 11-130 Translational Research Center, Philadelphia, PA, USA
Abstract Apolipoprotein (apo) E is a 299-residue protein which functions as a key regulator of plasma lipid levels. Human apoE exists as three common isoforms and the parent form, apoE3, operates optimally in promoting clearance of triglyceride (TG)rich lipoproteins and is associated with normal plasma lipid levels. This result occurs because apoE3 possesses both the requisite lipid-binding ability and affinity for the low density lipoprotein receptor (LDLR) to mediate appropriate lipolytic processing and endocytosis of TG-rich lipoprotein remnant particles. ApoE2 which differs from apoE3 by the single amino acid substitution Arg158Cys located near the LDLR recognition site exhibits impaired binding to the receptor and an inability to promote clearance of TG-rich lipoprotein remnant particles; this isoform is associated with Type-III hyperlipoproteinemia. ApoE4 which differs from apoE3 by the single amino acid substitution Cys112Arg is also associated with dyslipidemia although binding of this isoform to the LDLR is unaffected.
The amino acid substitution affects the organization and stability of both the N-terminal helix bundle domain and separately folded C-terminal domain so that apoE4 has enhanced lipid binding ability. As a consequence, apoE4 binds better than apoE3 to the surface of very low density lipoprotein (VLDL) particles and impairs their lipolytic processing in the circulation so that apoE4 is associated with a more pro-atherogenic lipoprotein-cholesterol distribution (higher VLDL-cholesterol/ high density lipoprotein-cholesterol ratio). This review summarizes current understanding of the structural differences between apoE2, apoE3, and apoE4, and the molecular mechanisms responsible for the alterations in lipoprotein metabolism resulting from this polymorphism of apoE. Detailed knowledge of how expression of structurally distinct apoE variants modifies lipoprotein metabolism provides a basis for developing C 2014 ways to manipulate the functionality of apoE in vivo. V IUBMB Life, 66(9):616–623, 2014
Keywords: apolipoprotein E; atherosclerosis; cholesterol; high density lipoprotein; lipoprotein metabolism; low density lipoprotein receptor; triglyceride; very low density lipoprotein
Introduction Apolipoprotein (apo) E is present on plasma lipoprotein particles in the circulation where it plays an important role in
Abbreviations: AAV, adeno-associated virus; Apo, apolipoprotein; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycan; LDLR, low density lipoprotein receptor; LPL, lipoprotein lipase; PL, phospholipids; TG, triglyceride; VLDL, very low density lipoprotein C 2014 International Union of Biochemistry and Molecular Biology V Volume 66, Number 9, September 2014, Pages 616–623 Address correspondence to: Michael Phillips, University of Pennsylvania, Philadelphia, Pennsylvania, USA. E-mail: [email protected] Received 11 August 2014; Accepted 9 September 2014 DOI 10.1002/iub.1314 Published online 9 October 2014 in Wiley Online Library (wileyonlinelibrary.com)
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cholesterol transport. This protein is a key regulator of plasma lipid levels, as exemplified by the situation in mice and humans lacking apoE where plasma cholesterol levels are markedly elevated and atherosclerosis is promoted (for a review, see (1)). The anti-atherogenic function of apoE arises in large part from its role in reducing plasma cholesterol levels by promoting clearance of triglyceride (TG)-rich lipoproteins from circulation (2,3). Human apoE exists as three common isoforms (apoE2, apoE3, and apoE4) and this polymorphism affects disease risk in carriers. The allele frequencies of e2, e3, and e4 in the human population are about 7, 78, and 14%, respectively (4). ApoE3 is considered to be the parent form and is associated with normal plasma cholesterol levels whereas the apoE2 and apoE4 isoforms have altered functionalities and are associated with the occurrence of hyperlipidemias. These abnormal plasma lipid levels and lipoprotein distributions are associated with increased risk of cardiovascular
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disease (1,2). This review summarizes current understanding of apoE structure-function relationships and of the molecular mechanisms responsible for the changes in the metabolism of TG-rich lipoproteins induced by the presence of the apoE2 and apoE4 isoforms.
Structure of Human ApoE3 Human apoE3 is a 299-residue molecule that contains multiple amphipathic a-helices which is a characteristic feature of exchangeable lipoproteins (5). The structure of apoE has been studied extensively over the past three decades (for reviews, see Refs. [6–8)). However, because of its flexible conformation and tendency to self-associate in aqueous solution, the highresolution structure of the entire apoE3 molecule was solved only recently (9). The NMR structure confirms earlier crystallographic studies in showing that the N-terminal region (residues 1–167) forms an anti-parallel four-helix bundle with the non-polar faces of the amphipathic helices facing the interior. This domain is separated by a hinge region from a C-terminal domain encompassing residues 206 to 299 that contains three a-helices which present a large exposed hydrophobic surface. These helices interact with those in the N-terminal helix bundle domain by formation of hydrogen-bonds and salt-bridges. Limited proteolysis studies (6) also have shown the existence of separately folded N- and C-terminal domains and this feature is indicated in Fig. 1. Protein engineering studies with apoE have demonstrated that the helix bundle domain contains a site recognized by the low density lipoprotein receptor (LDLR) and that the Cterminal domain is largely responsible for the lipid-binding ability of the protein (Fig. 1). The segment spanning residues 135 to 150 on helix four of the helix bundle domain is enriched in basic arginine (Arg) and lysine (Lys) residues that interact with acidic residues in the ligand-binding domain of members of the LDLR family (6). The Arg at position 172 is also critical for full LDLR binding activity (10). In addition to binding to acidic sites on the LDLR, the Arg and Lys residues in the apoE segment spanning residues 135 to 150 can also bind with high affinity to acidic sulfate groups on heparan sulfate proteoglycans (HSPG) (11). The specific positioning of the Arg and Lys residues in the amphipathic a-helix spanning residues 135 to 150 is important for maintaining optimal binding to the LDLR (12) but not for binding to HSPG (13). Studies of the lipid-binding and self-association properties of apoE3 variants containing C-terminal truncations indicate that the region spanning residues 260 to 299 is responsible for these properties of the apoE molecule (14–16). Lipid-free apoE in aqueous solution exists primarily as monomers, dimers, and tetramers and, consistent with the monomer being the form that can bind to lipid surfaces, the rate of dissociation of the oligomer to monomer is the rate-limiting step for lipidation (17). The exposed hydrophobic surface created by residues 260 to 270 in the C-terminal domain mediates the initial interaction of apoE with the surface of a phospholipid (PL)-contain-
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FIG 1
Structure of human apoE3. The 299-residue polypeptide chain is shown in linear form and the sites of the cysteine-arginine interchanges at positions 112 and 158 that distinguish apoE2 and apoE4 from apoE3 are marked. The protein is folded into two separate domains with N-terminal residues 1 to 191 containing an anti-parallel four-helix bundle and Cterminal residues 192 to 299 forming a separately folded domain that interacts with the helix bundle. The segment spanning residues 135 to 150 (horizontal red arrow) in the N-terminal domain contains a cluster of basic amino acids that form a binding site for the LDLR. The C-terminal segment spanning residues 260 to 299 (horizontal red arrow) contains amphipathic a-helices that initiate binding of the protein to lipid surfaces. See text for further details. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ing particle. After this first step involving insertion of the C-terminal amphipathic a-helices into the PL milieu at the particle surface, a second step ensues in which the N-terminal helix bundle opens to convert hydrophobic helix-helix interactions to helix-lipid interactions (18–20). In this step, helices one and two and helices three and four in the helix bundle preferentially remain paired (21). Furthermore, the open helix bundle adopts a partially extended conformation (22) and reorients with respect to the position of the C-terminal domain (23). Lipid association induces additional a-helix formation in apoE and when present in discoidal particles containing a segment of PL bilayer, the a-helices in both the N- and C-terminal domains are aligned perpendicular to the PL fatty acyl chains (24). Internal reflection infrared spectroscopy indicates that apoE interacts either with the edge of the PL bilayer or with the PL polar groups (25). The latter interaction occurs in quasi-spherical apoE/dipalmitoyl phosphatidylcholine particles where the two apoE molecules adopt a horseshoe shape (7,26). The conformation of lipid-bound apoE is dependent upon the shape and size of the particle with which it is associated. In the case of apoE bound to spherical lipid or lipoprotein particles, the N-terminal helix bundle can adopt either open or closed conformations (27,28). At low surface coverage, the four-helix bundle is open and all helices are in contact with lipid surface. In contrast, at high apoE surface concentrations, displacement of the N-terminal domain from the lipid surface by the more hydrophobic C-terminal domain causes the fourhelix bundle to adopt a closed conformation. This change in conformation alters the ability of apoE to bind to the LDLR because the helix bundle has to be open for recognition by the
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FIG 2
The role of apoE in the metabolism of plasma lipoproteins. Chylomicron particles secreted from the intestine and VLDL particles secreted from the liver are lipolyzed in the circulation by LPL to form remnant particles. ApoE (red) on such particles mediates their uptake in the liver by the LDLR or the LDLRrelated protein (LRP) and heparan sulfate proteoglycan (HSPG) pathways. The LDL particles formed by complete lipolysis of VLDL lack apoE and the apoB100 on such particles is the ligand for the LDLR. Adapted from Ref. 33. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
apo-B100 to the LDLR. ApoE is also important in the production of VLDL particles because its intracellular expression in hepatocytes promotes VLDL assembly and secretion (34). Optimal expression of apoE3 is crucial for normal metabolism of TG-rich lipoproteins. Over-expression and/or accumulation of apoE stimulates VLDL TG production which leads to hypertriglyceridemia. In addition, the presence of excess of apoE on VLDL particles can impair their lipolysis which elevates plasma TG levels (35). The lipid cargo delivered to hepatocytes by receptormediated endocytosis of TG-rich lipoprotein remnant particles is catabolized in lysosomes whereas the majority of the apoE remains in peripheral recycling endosomes. This pool of apoE is recycled back to the plasma membrane, resecreted and becomes part of an apoE-containing HDL pool (36). ApoE secreted by macrophages also contributes to the pool of apoEHDL. The expression of apoE in macrophages plays an important anti-atherogenic role by promoting cholesterol efflux from cells in the arterial wall (37).
ApoE Polymorphism receptor (6). Opening of the helix bundle alters the microenvironment in the receptor binding region around residues 135 to 150 so that there is greater positive electrostatic potential which enhances binding to acidic residues in the LDLR (29). In addition, lipid interaction also elongates the apoE helix in which the basic residues are situated, thereby enhancing the ability to bind to the LDLR (30). The open and closed conformations of the apoE helix bundle apparently coexist on the surface of hypertriglyceridemic very-low-density-lipoprotein (VLDL) particles because some of the apoE is recognized by the LDLR and some is not (31). Only the receptor-inactive conformation (helix bundle closed) of apoE is present at the surface of the VLDL particles isolated from normolipidemic individuals.
The polymorphism of human apoE is summarized in Table 1. The three major isoforms, apoE2, apoE3, and apoE4 differ by amino acid substitutions at positions 112 and 158. ApoE3 contains cysteine (Cys) at position 112 and Arg at position at 158, whereas apoE2 and apoE4 contains Cys and Arg, respectively, at both sites (6). ApoE3 is the most common isoform and is associated with normal lipoprotein metabolism whereas the less common apoE2 and apoE3 isoforms are associated with abnormal metabolism. ApoE also regulates lipid transport and cholesterol homeostasis in the brain and the apoE4 isoform is associated with Alzheimer’s disease (2,8). ApoE3 and apoE4 bind to the LDLR with similarly high affinity but the binding of apoE2 is some two orders of magnitude weaker (38).
Metabolic Functions of ApoE3
ApoE2 and Type III Hyperlipoproteinemia
ApoE in the peripheral circulation originates mostly from expression in hepatocytes, although there is a small contribution from expression in macrophages. Plasma apoE circulates associated with chylomicron, VLDL, and HDL particles where it plays a key role in their metabolism. As summarized in Fig. 2, chylomicrons derived from the intestine and VLDL derived from the liver are lipolyzed in the circulation by lipoprotein lipase (LPL) (32,33). ApoE on the remnant lipoprotein particles binds to the LDLR, LDLR-related protein and HSPG present on the surface of hepatocytes where the remnant particles are endocytosed and removed from the circulation. Some VLDL remnants are cleared rapidly whereas others undergo further lipolysis and are converted progressively to intermediate density lipoprotein (IDL) and finally to LDL. The LDL particles do not contain apoE and their clearance is mediated by binding of
The impaired binding of apoE2 to the LDLR is the reason for its association with Type III hyperlipoproteinemia. The degree to which receptor binding is reduced depends upon the nature of the lipoprotein particle to which apoE2 is attached and a mechanistic explanation for the defective binding has come from X-ray crystallographic comparisons of apoE2 and apoE3 (39). ApoE2 differs from apoE3 by the Arg to Cys interchange at position 158 which is close to the LDLR binding region (Fig. 1). This amino acid substitution in the amphipathic a-helix present in this region eliminates a salt-bridge between Arg158 and aspartic acid (Asp)154 which is present in apoE3 and leads to formation of a different salt-bridge between Asp158 and Arg150 in apoE2 (39). This new salt-bridge alters the conformation of Arg150 with respect to the other basic residues in the receptor-binding region so that it is no longer available to
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TABLE 1
Human apoE isoformsa
Protein
Apo E-2
Apo E-3
Apo E-4
Residue at position 112
Cysteine
Cysteine
Arginine
Residue at position 158
Cysteine
Arginine
Arginine
% LDL Receptor binding affinity
1
100
100
Population allelic frequency (%)
7
78
14
Type III Hyperlipoproteinemia
Normal
CVD and Alzheimer’s Disease
Clinical association a
Information taken from citations in the text.
interact with the LDLR, thereby reducing the ability of apoE2 to bind to the receptor. The impaired ability of apoE2 to bind to the LDLR leads to poor clearance of TG-rich lipoprotein remnants from the plasma compartment and predisposes carriers of this isoform to Type III hyperlipoproteinemia (33). The presence of apoE2 is essential but not sufficient to induce Type III hyperlipoproteinemia because, while this isoform does not bind to the LDLR well, its recognition by the LDLR-related receptor and HSPG is largely unaffected so that remnant lipoprotein clearance can still occur to some extent. Overt hyperlipidemia requires homozygosity for apoE2 combined with additional genetic or environmental factors such as obesity and a high fat diet to stress the system. Other mutations in or near the LDLR binding site of the apoE molecule which reduce the net positive electrostatic potential also give rise to Type III hyperlipoproteinemia (33,40). The severity of the hyperlipidemia associated with these rare apoE mutations is reflected in parallel reductions in the abilities of the isoforms to bind to cells and to heparin (41). Indeed, apoE variants that are defective in binding to all three types of cell surface receptor (LDLR, LDLR-related protein, HSPG) are associated with dominant inheritance of the trait and heterozygosity is sufficient for overt Type III hyperlipoproteinemia (33).
Comparison of the Structures and Properties of ApoE3 and ApoE4 While it is clear that the association of apoE2 with hyperlipidemia is a consequence of defective binding to the LDLR, this explanation does not account for the hypercholesterolemia associated with apoE4 (4) because this isoform binds like apoE3 to the receptor (38). The reason for the more proatherogenic lipoprotein-cholesterol distribution in the plasma of apoE4 carriers is the different lipoprotein distributions of apoE3 and apoE4 in plasma. When added to plasma, apoE3 binds preferentially to HDL and apoE4 binds more to VLDL
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(42,43). As summarized below, the molecular basis for this difference has been shown to be due to structural differences between the two apoE isoforms. The Cys112Arg substitution which converts apoE3 into apoE4 is located in the N-terminal helix bundle domain (Fig. 1) and replacement of the small Cys side chain with the bulky and positively charged Arg side chain destabilizes the helix bundle (44,45). The presence of Arg112 in apoE4 leads to rearrangement of the Arg61 side chain in the helix bundle, so that, unlike the situation apoE3, it forms a salt-bridge with glutamic acid (Glu) 255 in the C-terminal domain (46,47). This binding and probably other allosteric effects (48) lead to altered interactions between the N- and C-terminal domains in the apoE4 molecule. As a consequence, the C-terminal domain is organized differently in the two isoforms. For example, in apoE4 position 264 is less organized and more exposed to the aqueous environment (15) and the spacing between the N- and C-terminal domains is altered (49,50). Additionally, as assessed by urea denaturation, the apoE4 C-terminal domain is less stable and unfolds more readily than its counterpart in apoE3 (51). Overall, it is clear that the single amino acid substitution Cys112Arg significantly alters the structure of the protein so that the functions of apoE3 and apoE4 are different. Of particular note, the lipid binding properties of apoE3 and apoE4 are different with the latter isoform binding with higher affinity to lipid emulsion particles (52). Like apoE3, apoE4 binds to lipid particles by a two-step mechanism with the binding being initiated by the C-terminal domain (20). ApoE4 exhibits better lipid binding ability than apoE3 as a consequence, in part, of a rearrangement involving the segment spanning residues 260 to 272 in the C-terminal domain (Fig. 1) (16). The destabilization of the N-terminal helix bundle domain in apoE4 which allows it to unfold relatively readily compared to the situation in apoE3 also contributes importantly to the enhanced lipid binding ability of apoE4 (51). The strong lipid binding ability of apoE4 is the basis for the preferential binding of this isoform to VLDL particles, the surfaces of which are 60% PL-covered. ApoE4 binds much better than apoE3 to VLDL but somewhat less well than apoE3 to HDL.
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FIG 3
Contributions of different regions of the apoE3 and apoE4 molecules to their abilities to clear plasma cholesterol. The linear representations of the amino acid sequences show the position in the N-terminal helix bundle domain of the C112R (Cys112Arg) substitution that distinguishes the two isoforms. The fractional contribution to plasma cholesterollowering (F) is indicated beneath the indicated segments of the apoE3 and apoE4 molecules. The F values are calculated from the relative efficiencies of plasma cholesterol reduction for the apoE3 and apoE4 C-terminal truncation variants (1–272), (1–260), and (1–191) (57). For instance, the F value (0.8) of the segment spanning residues 261 to 272 in apoE3 is obtained by subtracting the cholesterol-lowering efficiency of apoE3 (1–260) (0.2) from that of apoE3 (1 to 272) (1.0). The importance of the segment spanning residues 261 to 272 is highlighted by the red bar marking this section of the apoE molecule. See text for further details. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Binding of apoE to HDL is mediated primarily by interaction of the N-terminal helix bundle domain with the resident apolipoproteins that cover 80% of the HDL particle surface. Thus, the selectivity in the binding of apoE3 and apoE4 to HDL and VLDL particles 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 particles, with the former being largely covered with PL and the latter with protein (16).
Comparison of the Influences of ApoE3 and ApoE4 on Lipoprotein Metabolism In Vivo The question of how the differences in structure and lipidbinding properties between apoE3 and apoE4 affect lipoprotein metabolism in vivo has proven difficult to answer. It is established that in mice expressing either human apoE3 or apoE4 in place of the endogenous mouse apoE, plasma total cholesterol levels are the same but expression of apoE4 is associated with higher VLDL-cholesterol and incidence of atherosclerosis (53,54). The strong anti-atherogenic function of apoE3 has been demonstrated by liver-specific expression
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using adeno-associated viral (AAV) vectors in apoE-deficient mice in which plasma cholesterol levels are reduced to normal wild-type mouse levels and atherosclerosis is prevented after 1 year of sustained expression (55). The clearance of apoEcontaining lipoproteins in mice occurs mainly by interaction with the LDLR (cf. Fig. 2) (56). With this background, we used the apoE-null mouse model and AAV to determine the effects on plasma cholesterol levels and lipoprotein-cholesterol distributions of expressing different doses of apoE3 and apoE4 and C-terminal deletion variants of both isoforms in the liver (57). In agreement with the findings in transgenic mice, apoE3, and apoE4 reduce plasma cholesterol levels to the same extent. As expected, the ability of apoE to bind to lipoprotein particles is critical for this effect because removal of the lipid-binding C-terminal domain greatly reduces effectiveness. Thus, expression of the isolated Nterminal domain (residues 1–191) of apoE3 gives zero cholesterol-lowering and about 30% activity in the case of apoE4 (Fig. 3). The fractional contributions to plasma cholesterol-lowering (F) of different regions of the apoE3 and apoE4 molecules are summarized in Fig. 3. It is apparent that residues 261 to 272 make the largest contribution to the reduction in plasma total cholesterol levels in both isoforms. This effect is consistent with the ability of apoE to bind to lipoprotein surfaces being critical for cholesterol-lowering because, as discussed earlier, this segment forms a hydrophobic surface patch which is key for lipid emulsion and VLDL binding activity. The different F value for residues 261 to 272 in apoE3 and apoE4 is consistent with the altered structural organization of this segment in the two proteins. The reduced F for this segment in apoE4 is compensated for by an increased contribution from its relatively unstable helix bundle domain (Fig. 3). Given that the overall plasma cholesterol-lowering ability of apoE4 is the same as that of apoE3, the question remains as to why apoE4 is associated with increased risk of cardiovascular disease. Insight into this issue was provided by an examination of the lipoprotein-cholesterol distributions in apoE3and apoE4-expressing mice (57). Importantly, under conditions where the plasma total cholesterol levels are the same apoE4 is associated with a more pro-atherogenic lipoprotein-cholesterol distribution (i.e. a higher VLDL-cholesterol/HDL-cholesterol ratio). This effect arises because VLDL processing via the LPL-mediated lipolysis cascade mentioned in Fig. 2 is relatively restricted in apoE4-expressing animals. The relative distribution of apoE between VLDL and HDL particles plays a crucial role in this process. The basis for the deranged behavior of apoE4 in the catabolism of VLDL is presented in detail in Fig. 4. In essence, the apoE content of VLDL has to be sufficient to support LDLR binding (and the apoE molecules have to be in the receptor-active conformation with the N-terminal helix bundle open) but not too much to displace apoC-II, the cofactor for LPL, and thereby inhibit lipolysis (33,58,59). The distribution of apoE3 between VLDL and HDL is appropriate for optimal progress down the lipolysis cascade. In contrast, the
Apolipoprotein E Isoforms and Lipoprotein Metabolism
FIG 4
Schematic comparing the influence of apoE3 and apoE4 on the lipolysis cascade involved in the catabolism of VLDL particles (57). After secretion from the liver into the plasma compartment, the triglyceride (TG) in VLDL is hydrolyzed by lipoprotein lipase (LPL) with apoC-II as a cofactor leading to the creation of intermediate density lipoprotein (IDL) and progressively smaller remnant particles. ApoE bound to these particles mediates their interaction with the LDLR and clearance from plasma (the lower red curved arrow shows the decrease in VLDL and remnant cholesterol levels). As the VLDL/IDL remnants shrink due to the removal of core TG, excess surface components (PL, cholesterol and apoE) are released into the HDL pool (upper blue curved arrow shows the increase in HDL cholesterol level). ApoE3 partitions between the VLDL and HDL pools so that lipolysis, clearance of VLDL remnant cholesterol and HDL formation is optimal. In the diagram, points c and d represent the VLDL/IDL-cholesterol and HDLcholesterol levels, respectively, when apoE3 is expressed. Relative to apoE3, apoE4 binds more to VLDL because of its higher lipid affinity leading to inhibition of lipolysis (probably because of displacement of apoC-II) so that, at the same apoE expression level, progression down the lipolysis cascade is relatively limited in the case of apoE4. The ratio a/b represents the apoE4 VLDL-C/HDL-C ratio which is higher than the apoE3 ratio c/d. See text for further details. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
greater lipid-binding ability of apoE4 increases the concentration of apoE4 on the VLDL surface so that lipolysis is impaired, resulting in reduced VLDL remnant clearance and an elevated VLDL-cholesterol/HDL-cholesterol ratio. The critical role for the ability of apoE to partition appropriately between VLDL and HDL during the lipolytic cascade is supported by the finding that deleting residues 273 to 299 to reduce the VLDL-binding affinity of apoE4 normalizes the VLDL-cholesterol/HDL-cholesterol ratio to that of apoE3 (57). This result suggests that pharmacologic intervention in apoE4 subjects to reduce the apoE content of VLDL could be beneficial therapeutically.
Summary and Conclusions The ability of apoE to bind to the surfaces of chylomicron, VLDL, and HDL particles as well as to cell surface molecules
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such as the LDLR and HSPG underlies its function as a key regulator of plasma cholesterol and TG levels. The dysfuctionality of apoE2 in lipoprotein metabolism arises from impaired binding to the LDLR whereas the dysfunctionality of apoE4 arises from enhanced binding to VLDL particles. The structural basis for these altered binding properties of these two isoforms is understood. The presence of Cys158 disrupts the nearby LDLR recognition site in the apoE2 molecule while the presence of Arg112 in apoE4 destabilizes the N- and Cterminal domains conferring enhanced lipid binding ability. Human apoE3 and apoE4 are equally effective at reducing total plasma cholesterol in apoE-null mice but the apoE4expressing animals have a pro-atherogenic lipoprotein profile (higher VLDL-cholesterol/HDL-cholesterol ratio) compared to their apoE3-expressing counterparts. Moderate reduction of the VLDL binding affinity of apoE4 reduces this proatherogenic lipoprotein profile.
Acknowledgements The author is indebted to all colleagues for their valuable contributions to the studies from this laboratory described here.
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