From the 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
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ABSTRACT |
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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.
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 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.
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
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 DH5 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 [ 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.
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).
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.
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.
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.
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).
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).
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.
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 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.
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-helices and contains the LDL
receptor binding region (residues 136-150 in helix 4) (17). The
amphipathic nature of the
-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
-helical
structure and contains the major lipid binding region (21, 22). The
detailed molecular features of the
-helices in the C-terminal domain
are poorly understood.
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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.
was
from Invitrogen. PCR supplies were from Qiagen (Chatsworth, CA).
Restriction enzymes were purchased from Promega (Madison, WI).
Isopropyl-
-D-galactopyranoside,
-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.
-13C]dimethyl lysine and
[
-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.
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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.
pKa values of lysine residues of full-length apoE3 in a
discoidal complex with DMPC
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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 (
) were
incubated with heparin-Sepharose gel for 2 h at room temperature.
Binding data of apoA-I in 50 mM NaCl buffer (
) 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.
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Fig. 3.
Binding isotherms of the apoE 10-kDa fragment
and variants to heparin. ( ) wild-type apoE 10-kDa fragment;
(
) 10-kDa(K233Q); (
) 10-kDa(K242Q); (
) 10-kDa(K262Q).
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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; (
) apoE3 22-kDa fragment; (
) apoE 10-kDa
fragment.
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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). ( ) apoE(K146Q); (
) apoE(K146E); (
)
apoE3(K233Q); (
) apoE(K233E). The data of wild-type apoE3
(dotted line) are also shown for comparison.
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Fig. 6.
Binding isotherms of apoE3 (1-260) and apoE
12-kDa fragment. ( ) apoE3 (1-260); (
) 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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-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
-helix. Consequently, they do not reduce the positive
electrostatic potential around the side chain of Lys-233.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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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.
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