Characterization of the Heparin Binding Sites in Human Apolipoprotein E*

Hiroyuki SaitoDagger §, Padmaja DhanasekaranDagger , David NguyenDagger , Faye BaldwinDagger , Karl H. Weisgraber, Suzanne WehrliDagger , Michael C. PhillipsDagger , and Sissel Lund-KatzDagger ||

From the Dagger  Joseph Stokes, Jr. Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4318 and the  Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, and Department of Pathology, University of California, San Francisco, California 94141

Received for publication, December 26, 2002, and in revised form, February 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the C-terminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pKa values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
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Apolipoprotein E (apoE)1 is a critical ligand for several hepatic lipoprotein receptors, including the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP), and for cell-surface heparan sulfate proteoglycans (HSPG) (1-3). Through its interaction with these receptors and with the HSPG-LRP pathway, apoE mediates the catabolism of remnant lipoproteins (4, 5). In the HSPG-LRP pathway, apoE is postulated to interact initially with cell-surface HSPG and then to transfer to the LRP for internalization (6, 7). Therefore, the interaction of apoE with HSPG is an initial step in the clearance of apoE-containing lipoproteins from the plasma. The apoE-HSPG interaction is also involved in the differential effects of the apoE isoforms on neurite outgrowth (8, 9), the existence of a pool of newly secreted apoE on the cell surface (10-12), and the inhibition of platelet-derived growth factor-stimulated smooth muscle cell proliferation (13). In addition, several studies suggest that binding of apoE to HSPG may be involved in Alzheimer's disease (5, 14, 15).

ApoE, a 299-amino acid, single chain protein, contains two independently folded functional domains, a 22-kDa N-terminal domain (residues 1-191) and a 10-kDa C-terminal domain (residues 222-299) (2, 16). The N-terminal domain exists in the lipid-free state as a four-helix bundle of amphipathic alpha -helices and contains the LDL receptor binding region (residues 136-150 in helix 4) (17). The amphipathic nature of the alpha -helix containing residues 136-150 is critical for normal binding to the LDL receptor (18). Our recent studies using nuclear magnetic resonance (NMR) demonstrated that Lys-143 and Lys-146 have unusually low pKa values because of local increases in positive electrostatic potential associated with the region surrounding residues 136-150 (19, 20). The C-terminal domain is also predicted to be a highly alpha -helical structure and contains the major lipid binding region (21, 22). The detailed molecular features of the alpha -helices in the C-terminal domain are poorly understood.

The N- and C-terminal domains each contain a heparin binding site (23, 24). The N-terminal domain site is located between residues 142-147 and overlaps with the receptor binding region (24). In fact, the HSPG binding activity of apoE variants is significantly decreased by mutations of Arg-142, Arg-145, and Lys-146, indicating that these basic amino acid residues contribute to heparin binding (25). A recent study on the interaction between a heparin-derived oligosaccharide and the N-terminal domain of apoE4 found that Arg-142 and -145 form salt bridges with sulfate groups from the octasaccharide fragment (26), consistent with a dramatically reduced binding affinity of apoE4(R142C) and apoE4(R145C) mutants for HSPG (25). This study also predicted additional interactions between the heparin fragment and Lys-143, Lys-146, and Arg-147. Most recently, a site-directed mutagenesis study showed that Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 are required for high-affinity binding to heparin, with Lys-146 participating in an ionic interaction with the heparin fragment and Lys-143 participating in a hydrogen bond (27).

The goal of the current study was to identify the heparin binding site in the C-terminal domain of apoE and to characterize its contribution to the apoE-heparin interaction in both lipid-free and lipidated states. We demonstrated that the isolated C-terminal domain binds to heparin with higher affinity than the N-terminal domain in the lipid-free state and that an unusual basic microenvironment around Lys-233 is involved in heparin binding. Surprisingly, this C-terminal site is unavailable for heparin binding in both the lipid-free and lipidated states of the intact apoE molecule, suggesting that only the N-terminal site contributes to the interaction of apoE with HSPG in vivo.

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Materials-- 1,2-dimyristoyl phosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Pelham, AL), and stock solutions were stored in chloroform/methanol (2/1) under nitrogen at -20 °C. Its purity was assayed by thin-layer chromatography on silica gel G plates (Analtech, Newark, DE) in chloroform/methanol/water (65/25/4). Lipids were visualized by spraying developed thin-layer plates with a 50% sulfuric acid solution and charring at 200 °C for 15 min; 100-µg quantities gave a single spot by charring. Heparin-Sepharose CL-6B (Lot No. S.E. 203810) was purchased from Amersham Biosciences. Ligand density, determined using an enzymatic kit for heparin from DiaPharma (West Chester, OH), was ~3 mg of heparin/g dried gel. Sepharose CL-6B was from Sigma. D2O (Cambridge Isotope Laboratories, Andover, MA) was routinely deoxygenated and stored under nitrogen. [13C]Formaldehyde (99% isotopic enrichment) as a 20% solution in water was also obtained from Cambridge Isotope Laboratories. [14C]Formaldehyde (40-60 Ci/mol) in distilled water was purchased from PerkinElmer Life Sciences. NaCNBH3 (Aldrich, Milwaukee, WI) was recrystallized from methylene chloride before use. All other salts and reagents were analytical grade.

Bacteriological media were obtained from Fisher (Pittsburgh, PA). The prokaryotic expression vector pET32a was from Novagen (Madison, WI), and the competent Escherichia coli strain BL21 star (DE3) was from Invitrogen. Competent E. coli strain DH5alpha was from Invitrogen. PCR supplies were from Qiagen (Chatsworth, CA). Restriction enzymes were purchased from Promega (Madison, WI). Isopropyl-beta -D-galactopyranoside, beta -mercaptoethanol, aprotinin, and ampicillin were from Sigma. Ultrapure guanidine-HCl was from ICN Pharmaceuticals (Costa Mesa, CA). Oligonucleotides were from IDT (Coraville, IA), and DNA purification kits were from Qiagen.

Expression and Purification of Proteins-- The mutations of K146Q, K146E, K233Q, and K233E in full-length apoE3 and K233Q, K242Q, and K262Q in the 10-kDa fragment were introduced by using PCR to create DNA inserts that were ligated into a thioredoxin fusion expression vector (pET32a), as described (18, 28). The mutation, sequence, and cDNA orientation were confirmed by restriction enzyme analysis and double-stranded DNA sequencing. The resulting fusion proteins were expressed in E. coli, cleaved, and purified as described (28). C-terminal-truncated apoE3, apoE3 (1-260), full-length human apoE3, and its 22- and 10-kDa fragments were expressed and purified as described (29). The 12-kDa fragment of apoE3 (residues 192-299) was prepared by thrombin digestion of full-length apoE3 (30).

NMR Measurements-- Full-length apoE3 was complexed with DMPC and isolated by gel-filtration chromatography (19). The lysine residues in the apoE3-DMPC complexes were labeled with 13C by reductive methylation (31). 1H-13C heteronuclear single quantum coherence (HSQC) two-dimensional NMR spectra of 13C-labeled full-length apoE3-DMPC complexes were obtained with a Bruker DM×600 wide-bore spectrometer equipped with an SGI 02 computer and a 5-mm inverse broadband probe (19). The two-dimensional 1H-13C HSQC spectra were recorded with carbon decoupling during acquisition at 310 K. The time-proportional phase-increment method was used to obtain phase-sensitive spectra. Chemical shifts and line widths for lipid-protein complexes were measured as described (19, 31, 32). The chemical shifts of [epsilon -13C]dimethyl lysine and [epsilon -13C]dimethyl terminal amino residues of the complexes were determined as a function of pH. The pKa values of the [13C]dimethyl lysines were obtained by nonlinear regression fitting of the chemical shifts at different pH values to the Henderson-Hasselbalch equation with the GraphPad Prism program (GraphPad Software, San Diego, CA) (19, 20). The sigmoidal equation is Y = (U + W × 10(X-Xc))/(10(X-Xc) + 1) where Y is the chemical shift, U is the lower limit of the shift, W is the upper limit, X is pH, and Xc is pKa.

Binding of ApoE to Heparin-- The binding of apoE to heparin was assayed using a centrifugation method (24, 33). ApoE samples were 14C-labeled to a specific activity of ~1 µCi/mg protein by reductive methylation of lysines with [14C]formaldehyde as described (19, 29, 31). 14C-labeled lipid-free apoE or apoE-DMPC complexes were incubated with heparin-Sepharose or Sepharose gel (0.3 mg of gel/ml) for 2 h at room temperature in 300 µl of Tris buffer (10 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3, 1 mM EDTA, pH 7.4). The mixture was then centrifuged at 10,000 rpm for 15 min in a tabletop centrifuge. The radioactivity in 200-µl aliquots of the top fraction and in 100-µl aliquots of the bottom fraction was quantitated, and the amount of apoE bound to the gel was calculated by subtracting the background-free apoE concentration in the bottom fraction. Heparin-bound apoE expressed in g of apoE bound per g of dried weight (the gel was dried by incubation for 24 h at room temperature in a vacuum oven) of gel was corrected for any binding to the Sepharose itself by subtraction of Sepharose gel-bound radioactivity obtained in a parallel incubation. Binding data were fitted by nonlinear regression to a one-binding site model by using the GraphPad Prizm program.

Analytical Procedures-- Protein concentrations were determined by the Lowry procedure (34). Phospholipid content was monitored by phosphorus analysis (35). 14C radioactivity was assessed by standard liquid scintillation procedures. SDS-PAGE (8-25% gradient) was performed with a Pharmacia Phast electrophoresis system to monitor the purity of the proteins.

    RESULTS
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pKa Values of Lysines in Full-length ApoE3-- Previously, we used two-dimensional NMR spectra of [13C]dimethyl lysines to study the microenvironments of the eight lysine residues in 22-kDa apoE-DMPC complexes (19, 20). In this study, application of a higher field (600 MHz) NMR spectrometer enabled us to evaluate the microenvironments of all 12 lysine residues of full-length apoE3 because of the enhanced resolution of the resonances in the NMR spectrum. Fig. 1 shows an NMR spectrum and the sequence-specific assignment of the 12 [13C]dimethyl lysine resonances in full-length apoE3-DMPC discoidal complexes. In addition to the eight lysine resonances in the N-terminal domain, four lysine resonances in the C-terminal domain (Lys-233, -242, -262, and -282) were resolved and assigned using a selective lysine-to-glutamine mutation technique (19).


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Fig. 1.   Phase-sensitive 600-MHz 1H-13C HSQC NMR spectrum of full-length apoE3-DMPC discoidal complexes at 37 °C. All the lysine residues were converted to [13CH3]2 lysine by reductive methylation. The 2.5/1 w/w DMPC/full-length apoE3 discoidal complexes (3.2 mg of protein/ml) were dissolved in borate buffer (pH 10.1). Spectra were obtained under conditions similar to those used previously (19). The assignments for the 12 lysine resonances are indicated.

The pKa value for each lysine was obtained by monitoring the chemical shift as a function of pH over a range of pH values (5.5-12.5). The titration curves were fully reversible across the pH range studied (data not shown), and the pKa values for all the lysines in the full-length apoE3 are listed in Table I. As expected, the pKa values for eight lysines in the N-terminal domain of full-length apoE3 were the same as those for the 22-kDa fragment of apoE3 reported previously (19). Lys-233 has a low pKa of 9.4, which is similar to the pKa values of Lys-143 and -146 located in the LDL receptor/heparin binding region in the N-terminal domain of apoE3. This suggests that Lys-233 may behave similarly and be part of the heparin binding region in the C-terminal domain.


                              
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Table I
pKa values of lysine residues of full-length apoE3 in a discoidal complex with DMPC

Heparin Binding of the 10-kDa Fragment-- To determine the relative heparin binding activities of apoE molecules, we measured the binding of apoE to heparin-Sepharose gel (24, 33). The binding of the 10-kDa fragment to heparin was saturable and depended on the salt concentration (Fig. 2). The dissociation constant (Kd) was calculated to be 26 µg/ml at 50 mM NaCl and 76 µg/ml at 150 mM NaCl, consistent with a previous report (24). The maximal binding capacities at both concentrations were similar and corresponded to about 140-170 g of protein/g heparin. In contrast, apoA-I did not bind to heparin even at low salt concentration. These results indicate that the interaction between the 10-kDa fragment and heparin is electrostatic (36, 37) and the amphipathic characteristics common in helical apolipoproteins (38) are not responsible for heparin binding.


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Fig. 2.   Effect of salt concentration on apolipoprotein binding to heparin. Varying concentrations of the apoE 10-kDa fragment in Tris buffer, pH 7.4, containing 50 mM NaCl () or 150 mM NaCl (open circle ) were incubated with heparin-Sepharose gel for 2 h at room temperature. Binding data of apoA-I in 50 mM NaCl buffer (black-square) are also shown. Each point represents the mean ± S.D. from two independent experiments, each done in duplicate. The binding curves were obtained by nonlinear regression fitting to a one-binding site model.

The basic residues (arginine and lysine) in the heparin binding region of the N-terminal domain are key to the interaction with heparin (26, 27, 39). We compared the heparin binding activities of the three lysine mutants (K233Q, K242Q, and K262Q) of the 10-kDa fragment to identify the heparin binding site in the C-terminal domain of apoE. As shown in Fig. 3, a large decrease in heparin binding was observed only for the 10-kDa (K233Q) variant (maximal binding capacity was 31% of the wild-type 10-kDa fragment) without a change in binding affinity (Kd values for both were 76 µg/ml). Of the four lysines in the C-terminal domain, only Lys-233 showed a relatively low pKa value (Table I), suggesting that an unusually basic microenvironment around Lys-233 is critical for the interaction between the C-terminal domain and heparin.


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Fig. 3.   Binding isotherms of the apoE 10-kDa fragment and variants to heparin. (open circle ) wild-type apoE 10-kDa fragment; () 10-kDa(K233Q); (black-triangle) 10-kDa(K242Q); (down-triangle) 10-kDa(K262Q).

Comparison of Heparin Binding of Full-length ApoE3 and Its 22- and 10-kDa Fragments-- To determine the relative contributions of the N- and C-terminal domains to the heparin binding of apoE, we next compared the heparin binding of full-length apoE3 and its 22- and 10-kDa fragments in both lipid-free and lipidated forms. In the lipid-free form, full-length apoE and the 10-kDa fragment bound to heparin with similar affinities, whereas the 22-kDa fragment bound to heparin with much lower affinity (Fig. 4A). When complexed with DMPC, the heparin binding of the 10-kDa fragment was completely abolished, indicating that the C-terminal binding site is masked by lipids (Fig. 4B). Instead, the lipidated 22-kDa fragment bound to heparin with high affinity and rather better than full-length apoE. Similar results were obtained with POPC, a more physiological phospholipid (data not shown). Although the binding capacity of lipidated full-length apoE3 was much lower than that of the lipid-free form, the binding affinity of full-length apoE3 in both states was similar (Kd for the lipid-free and lipidated forms were 157 and 210 µg/ml, respectively), consistent with the previous observation using surface plasmon resonance analysis (37).


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Fig. 4.   Binding isotherms of full-length apoE3 and its 22- and 10-kDa fragments to heparin in the lipid-free form (A) and complexed with DMPC (B). () full-length apoE3; (black-triangle) apoE3 22-kDa fragment; () apoE 10-kDa fragment.

Heparin Binding of ApoE Mutants-- The above observations suggest that the two apoE domains contribute differently to heparin binding, with the C-terminal domain playing a dominant role in the lipid-free state and the N-terminal domain in the lipidated state. To examine this hypothesis, we introduced mutations into two lysines of full-length apoE and compared the heparin binding activities of these variants. The mutated proteins were apoE(K146Q) and apoE(K146E), which have N-terminal domains that bind defectively to heparin (27), and apoE3(K233Q) and apoE3(K233E), which are thought to have low heparin binding capacity through the C-terminal site. Large decreases in heparin binding were observed with apoE(K146E) in both the lipid-free (Fig. 5A) and the DMPC-complexed forms (Fig. 5B), indicating that Lys-146 contributes greatly to the heparin binding of full-length apoE. However, as also shown in Fig. 5, A and B, the lipid-free apoE(K146Q) exhibited a much smaller decrease in heparin binding than wild-type apoE3, and apoE(K146Q)-DMPC complexes bound to heparin in a similar manner to the lipid-free form. This disparity between apoE(K146E) and apoE(K146Q) variants is probably because of the ionization state of the glutamate residue at pH 7.4 that is responsible for the difference in the heparin binding activities (27).


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Fig. 5.   Binding isotherms of full-length apoE variants to heparin in the lipid-free form (A and C) and complexed with DMPC (B and D). (triangle ) apoE(K146Q); () apoE(K146E); () apoE3(K233Q); (black-down-triangle ) apoE(K233E). The data of wild-type apoE3 (dotted line) are also shown for comparison.

In contrast, the K233Q mutation had no effect on the heparin binding activity of either the lipid-free or the lipidated form. In the case of the apoE3(K233E) variant, a small decrease in heparin binding was observed in the lipid-free form; complexing with DMPC had virtually no effect on the heparin binding ability of this variant (Fig. 5, C and D), suggesting that the Lys-233 site is unavailable for heparin binding of full-length apoE. For wild-type apoE and the apoE(K146E) and apoE(K233Q) variants, the heparin binding of free protein differs from that of the protein-DMPC complexes. Binding to DMPC results in the opening of the four-helix bundle in the N-terminal domain of apoE (40-42). Therefore, it is likely that these apoE variants have subtle differences in their final conformations on the DMPC disc that may modulate their heparin binding properties (27). This may be the reason why the lipidated K146Q and K223E variants bind somewhat more to heparin than wild-type apoE3.

To further explore the role of the C-terminal domain in the heparin binding of apoE, we used apoE (1-260), a C-terminal truncated variant that is monomeric rather than tetrameric in aqueous solution (21). Although the C-terminal heparin binding site is conserved in this variant, the heparin binding of apoE (1-260) was significantly lower than wild-type apoE3 and close to that of the apoE3 22-kDa fragment (Fig. 6). This indicates that tetramerization through the C-terminal domain has a major influence on the heparin binding ability of the lipid-free apoE. We also measured the binding of the 12-kDa apoE fragment (2) to heparin to investigate whether the hinge region plays a role in the heparin interaction of the C-terminal domain. Surprisingly, the heparin binding ability almost disappeared for the lipid-free 12-kDa fragment (Fig. 6), suggesting that the hinge region may mask the heparin binding site of the C-terminal domain.


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Fig. 6.   Binding isotherms of apoE3 (1-260) and apoE 12-kDa fragment. (open circle ) apoE3 (1-260); (black-square) 12-kDa fragment. The data of wild-type apoE3 (dotted line) and apoE3 22-kDa fragment (dashed line) are also shown for comparison.


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The interaction between apoE and HSPG is important in several processes, such as lipoprotein remnant clearance (5, 43) and neurite outgrowth (3, 14). ApoE has at least two heparin binding sites located between residues 142 and 147 in the N-terminal receptor binding region (24) and less well defined sites in the C-terminal lipid binding region (23, 24). The N-terminal site has recently been characterized using apoE4 22-kDa fragments and an enzymatically prepared heparin oligosaccharide; Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 within this site are involved in the interaction with the heparin fragment (27). In this study, using site-directed mutagenesis we identified Lys-233 as a key lysine residue in the C-terminal site and also estimated the relative contributions of both sites to the heparin binding of full-length apoE. Earlier studies using either synthetic apoE peptides or blocking monoclonal antibodies suggested that residues 211-218 (23) and 243-272 (24) contain heparin binding sites. The former site is not a factor in the observed binding of the C-terminal 10-kDa fragment to heparin because it is not included in this domain. It is also not functional in the 12-kDa fragment because this does not bind to heparin (Fig. 6). Presumably, the small peptide containing residues 211-218 (23) can bind because its conformation is different from when it is present as part of the apoE molecule. It is possible that Lys-233 is part of the heparin binding site identified by a blocking antibody with an epitope located between residues 243-272 (24); the presence of the antibody may interfere with the functionality of Lys-233.

Our NMR results imply that among all 12 lysines of full-length apoE3, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pKa values, indicative of high positive electrostatic potential around these residues. Previous studies showed that this greater positive electrostatic potential around Lys-143 and -146 is involved in the high-affinity binding of the N-terminal domain of apoE to the LDL receptor and heparin. In this study, we demonstrated that residue Lys-233 is critical for the heparin binding of the C-terminal domain (Fig. 3), indicating that an unusually basic lysine microenvironment in the C-terminal domain is also involved in the heparin interaction. In an amphipathic alpha -helix spanning this region, the basic residues Arg-226, Lys-233, and Lys-240 are aligned, two turns apart, along one side of the helix. It is interesting that there are several acidic residues in the amino acid sequence near Lys-233, but they are mostly located on the opposite side of the amphipathic alpha -helix. Consequently, they do not reduce the positive electrostatic potential around the side chain of Lys-233.

Comparison of the heparin binding abilities of full-length apoE3 and its 22- and 10-kDa fragments yields interesting insights. In the lipid-free state, the 10-kDa fragment binds to heparin with higher affinity than the 22-kDa fragment (Fig. 4A). In contrast, the heparin binding of the 10-kDa fragment was completely abolished when complexed with lipid (Fig. 4B), consistent with previous observations showing that the C-terminal site is only available for interaction in the lipid-free state (24, 44). The C-terminal domain of apoE forms a stable tetramer and is responsible for self-association of apoE in aqueous solution, whereas the N-terminal domain is primarily monomeric (16). Therefore, the stronger interaction of the C-terminal domain with heparin appears to be because of its tetramerization. As shown in Fig. 6, the heparin binding affinity of the apoE3 (1-260) variant, which is monomeric in solution, is much lower than that of wild-type apoE3 and is comparable with that of the 22-kDa fragment, indicating that tetramerization through the C-terminal domain is crucial for the heparin binding ability of the lipid-free apoE. Multiple sequences of heparin binding peptide enhance heparin binding through cooperativity effects (45). Similarly, multiple copies of the receptor binding domain are necessary for the high affinity interaction of apoE with the LDL receptor (46, 47). In this regard, the different binding affinities of full-length apoE3 and the 22-kDa fragment in DMPC discs (Fig. 4B) may be explained by the difference in cooperativity arising from different numbers of the N-terminal domain per particle.

Despite the strong heparin binding ability of the 10-kDa fragment, the C-terminal site of full-length apoE3 does not appear to be involved in the heparin binding of lipid-free apoE. As shown in Fig. 5C, there were only small differences in heparin binding among the lipid-free wild-type apoE3, apoE3(K233Q), and apoE3(K233E) variants, suggesting that, even in the lipid-free state, only the N-terminal site is involved in the heparin interaction of full-length apoE. It is not clear why the C-terminal site of the lipid-free apoE is unavailable to interaction with heparin. One possible explanation is that the heparin interaction of the C-terminal domain is sterically hindered by the hinge region (residues 192-215), which acts as a spacer connecting the two domains. Indeed, the 12-kDa fragment, which contains both the 10-kDa domain and the hinge region, bound defectively to heparin (Fig. 6). Thus, interaction between the 10-kDa domain and the hinge region might mask the C-terminal heparin binding site (the basic residues around Lys-233), preventing interaction with heparin. A recent fluorescence resonance energy transfer study of apoE3 indicates spatial proximity between the N- and the C-terminal domains in the lipid-free state, with the hinge region tethering the N-terminal domain close to the C-terminal domain (48).

ApoE-containing high density lipoproteins bind to the proteoglycan, biglycan, in arterial walls, and apoE is thought to act as a bridging molecule that traps these particles in atherosclerotic intima (49). This binding interaction between apoE and biglycan was shown recently to occur via the C-terminal domain in which the charged residues, especially lysine and arginine between residues 223 and 230, are involved in the binding (50, 51). The results of the present study suggest that the unusually basic microenvironment around Lys-233 contributes to the apoE-biglycan interaction. Recent studies demonstrated that C-terminal fragments of apoE are complexed with amyloid in the brain (52, 53) and that C-terminal-truncated forms of apoE occur in Alzheimer's disease (54). The presence of apoE proteolytic fragments in vivo raises the possibility that the C-terminal fragment may contribute to Alzheimer's disease by interaction with HSPG in the brain.

In conclusion, we have identified the heparin binding site of the C-terminal domain of apoE. Our data show that the basic residues around lysine 233 are critical for the heparin binding of the C-terminal domain but are unavailable for heparin binding in full-length apoE even in the lipid-free state. Thus, it seems that lipid-poor apoE associates with cell surface HSPG only through the N-terminal domain, leaving the C-terminal domain available for binding to lipoprotein particles.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL56083 (to S. L. K.) and HL41633 (to K. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: National Institute of Health Sciences, 1-1-43 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan.

|| To whom correspondence should be addressed: Joseph Stokes, Jr. Research Inst., The Children's Hospital of Philadelphia, Abramson Research Bldg., Suite 302, 3615 Civic Center Blvd., Philadelphia, PA 19104-4318. Tel.: 215-590-0588; Fax: 215-590-0583; E-mail: katzs@ email.chop.edu.

Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M213207200

    ABBREVIATIONS

The abbreviations used are: apo, apolipoprotein; HSPG, heparan sulfate proteoglycan; HSQC, heteronuclear single quantum coherence; LDL, low density lipoprotein; LRP, LDL receptor-related protein; DMPC, 1,2-dimyristoyl phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; NMR, nuclear magnetic resonance.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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