© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Biglycan, a Vascular Proteoglycan, Binds Differently to HDL2 and HDL3
Role of ApoE
From the Departments of Medicine (K.L.O., A.C.) and Pathology (S.P.-P., T.N.W.), University of Washington, Seattle, and the Department of Medicine (H.R.B.), University of Western Australia, Perth.
Correspondence to Dr Alan Chait, Box 356426, Department of Medicine, University of Washington, Seattle, WA 98195-6426. E-mail achait{at}u.washington.edu
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Abstract |
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Abstract—Lipoprotein retention by vascular extracellular matrix proteoglycans is important in atherogenesis. Proteoglycans bind apolipoprotein (apo)B- and apoE-containing lipoproteins. However, the colocalization of apoA-I and apoE with biglycan in atherosclerotic lesions suggests that vascular proteoglycans also may trap high density lipoproteins (HDLs). Because the major HDL subclasses may be atheroprotective to different degrees, we investigated the role of apoE in mediating HDL2 and HDL3 binding to the extracellular vascular proteoglycan, biglycan. ApoE-free HDL2 and HDL3 did not bind to purified [35S]SO4-biglycan, whereas apoE-containing HDL2 and HDL3 (HDL+E) did. The extent of binding correlated positively with the apoE content for both HDL2 and HDL3, although HDL2+E had a 3.5-fold higher affinity than did HDL3+E. ApoE on HDL3 was cleaved into 22- and 12-kDa fragments, whereas apoE on HDL2 remained intact. These results suggest that the cleaved apoE on HDL3 results in diminished biglycan binding of HDL3+E relative to HDL2+E. Reducing positive charges on lysine and arginine residues on HDL+E eliminated biglycan binding, suggesting an ionic interaction. Thus, apoE is an important determinant of HDL binding to extracellular vascular proteoglycans and may play a role in HDL retention in the artery wall.
Key Words: high density lipoproteins • biglycan • atherosclerosis • proteoglycans • apolipoprotein E
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Introduction |
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Retention of lipoproteins by extracellular matrix molecules is believed to be critical in the pathogenesis of atherosclerosis.1 2 3 One class of matrix molecules, proteoglycans, is well known for its ability to retain lipoproteins in the arterial wall by interaction of negatively charged groups on the proteoglycan side chains with clusters of positively charged amino acid residues on apoB and apoE.4 5 Major extracellular proteoglycans found in human coronary artery atherosclerotic lesions include the small dermatan sulfate proteoglycan, biglycan,6 7 8 which is synthesized by vascular smooth muscle cells.9 10 11 12 Recent data suggest that biglycan plays an especially significant role in the trapping and retention of lipoproteins because it was found to colocalize with apoB and apoE in human atherosclerotic lesions.8 13 HDLs are believed to be antiatherogenic.14 15 However, apoA-I, the major apolipoprotein of HDL, is seen in atherosclerotic lesions in humans,8 16 17 18 19 20 colocalizing with biglycan, apoB, and apoE.8 It also is found extracellularly in atherosclerotic lesions in mice.21 We previously have shown that apoE-free HDL3 (HDL3-E) did not bind to purified extracellular proteoglycans but that apoE-containing HDL (HDL+E) bound to vascular proteoglycans with relatively high affinity.8 Because apoE-free HDL (HDL-E) contains a high content of apoA-I, this strongly suggests that apoA-I does not bind to proteoglycans. Furthermore, it has been reported that mammalian apoA-I does not contain heparin-binding domains.22 Because HDL-E does not bind to proteoglycans, the observation that apoE and apoA-I, the major apolipoprotein of HDL, colocalize with biglycan in human coronary artery lesions raises the question of whether the retention of certain HDL subclasses by extracellular proteoglycans may be mediated in part by apoE. Therefore, we have studied the role of apoE in mediating the binding in vitro of the 2 major subclasses of HDL, HDL2 and HDL3, to the extracellular vascular proteoglycan, biglycan.
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Methods |
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Lipoproteins
HDL2 (density 1.063 to 1.125 g/mL), HDL3 (density 1.125 to 1.21 g/mL), and LDL (density 1.019 to 1.063 g/mL) were isolated by sequential density ultracentrifugation from plasma obtained from a pool of healthy human volunteers, as described previously.23 The HDL fractions were dialyzed extensively against 50 mmol/L imidazole buffer (pH 6.7) at 4°C and concentrated (Centriprep 100, Amicon). HDL-E and HDL+E fractions were prepared by passing the HDL fractions over a heparin-Sepharose column that had been equilibrated with 50 mmol/L imidazole buffer. The unbound (apoE-free) fractions, HDL2-E and HDL3-E, were collected with a fraction collector, and those fractions that contained the highest concentrations of cholesterol were pooled. The bound (apoE-containing) HDL fractions, HDL2+E and HDL3+E, were eluted off the column with 1 mol/L NaCl. The HDL fractions and LDL were dialyzed extensively against 150 mmol/L NaCl/1 mmol/L EDTA (pH 7.4) at 4°C. Lipoproteins were stored in the presence of nitrogen at 4°C in the dark and used within 3 weeks of preparation.
To minimize proteolytic and oxidative degradation of apolipoproteins ex vivo, samples were kept on ice at all times, and protease inhibitors were present throughout the lipoprotein isolation procedure. Sodium azide (final concentration 125 µmol/L, Sigma Chemical Co), EDTA (final concentration 1 mmol/L), and soybean trypsin inhibitor (final concentration 200 µg/mL, Sigma) were added to whole blood immediately after the blood was drawn; phenylmethylsulfonyl fluoride (final concentration 10 µmol/L, Sigma) and Phe-Pro-Arg chloromethyl ketone (PPACK) dihydrochloride (final concentration 1 µg/mL, Calbiochem) were added to plasma immediately after separation from red blood cells. Solutions used during lipoprotein isolation contained 1 mmol/L EDTA to prevent oxidative modification.
Because the HDL2 fraction usually contained a small amount of apoB, apoB-containing particles were selectively removed by using an immunomagnetic procedure, according to the manufacturer’s instructions. An affinity-purified polyclonal anti-human apoB100 antibody (Academy Bio-Medical Company, Inc) was bound to Dynabeads (M-280, Dynal, Inc), which was mixed with HDL2+E overnight at 4°C. After 1 minute in a magnet, the supernatant was removed and analyzed for apoA-I, apoB, and apoE, as described below.
Lipoprotein triglyceride, phospholipid, and cholesterol content were determined enzymatically by using an Abbott Spectrum Multichromatic Analyzer. Total protein content was measured by the Lowry method.24 Apolipoprotein content (apoE, apoA-I, and apoA-II) was quantified by immunonephelometry (Behring Nephelometer Analyzer).
The presence or absence of apoB and apoE in the LDL and HDL preparations was evaluated by SDS-PAGE and Western blotting. Samples were electrophoresed on 7% to 17% polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and probed with either a mouse monoclonal antibody against apoB (MB-47, titer 1:2000; a kind gift from Dr Linda Curtiss, Scripps Research Institute, La Jolla, Calif) or an affinity-purified goat polyclonal antibody against apoE (titer 1:1000; a kind gift from Dr John Albers, University of Washington, Seattle). (See Supplementary Online Information, which can be found in a data supplement available at http://atvb.ahajournals.org.)
To ensure that the cleavage of apoE did not affect the
accuracy of its quantification by immunonephelometry, apoE on HDL was
systematically cleaved in the presence of
thrombin25 and then subjected
to immunonephelometry for apoE quantification. Briefly,
HDL2+E was incubated with 50 U of 
-thrombin
(a kind gift from Dr Sandra Gianturco, University of Alabama at
Birmingham) per milligram protein for 1 hour, 2 hours, or 4 hours at
37°C, followed by the addition of the potent thrombin
inhibitor PPACK (4:3, PPACK:thrombin). Samples were stored
at 4°C and analyzed within 24 hours for apoE content by
immunonephelometry, as described above.
HDL Modifications
The interaction of lipoproteins with proteoglycans is
thought to be primarily ionic in nature. Thus, the role of positively
charged amino acids on apoE of HDL was investigated by neutralizing
arginine and lysine residues with the use of cyclohexanedione and
acetylation, respectively, as described
previously26 (see
Supplementary Online Information). Immediately after these modification
procedures, HDL was dialyzed extensively into HEPES sample buffer,
containing 25 µmol/L butylated hydroxytoluene, at 4°C. The modified
HDL samples, compared with native HDL, were then analyzed for
their ability to bind to biglycan. The extent of derivatization was
assessed by trinitrobenzenesulfonic acid reactivity, which measures
free amino groups, as described
previously,27 or by amino
acid
analysis.28
Biglycan Isolation
[35S]SO4-labeled
biglycan synthesized by cultured human aortic smooth muscle cells was
isolated as described
previously29 (see
Supplementary Online Information). The
glycosaminoglycan content of the
[35S]SO4-labeled
biglycan was quantified by using dimethyl methylene blue as described
previously.30 Calculated
molarities were based on the assumption that biglycan contained 2
chains each of 59
kDa.10 11 31
Therefore, we estimated that 1 µmol/L
[35S]SO4-biglycan
was 
118 µg/mL glycosaminoglycan. The purity of
biglycan was confirmed by Western blot
analysis29 with use
of an antibody specific for biglycan (LF-51; a gift from Dr Larry
Fisher, Bone Research Branch, National Institute of Dental Research,
National Institutes of Health, Bethesda,
Md)32 and enhanced
chemiluminescence (Western-Light Chemiluminescent Detection System with
CSPD substrate, Tropix).
Gel Mobility Shift Assay
The interaction of the lipoprotein preparations with
purified arterial biglycan was assessed by using an
electrophoretic gel mobility shift assay described by Camejo,
Hurt-Camejo, and
colleagues.33 34
The amount of complexed versus free
[35S]SO4-biglycan
in each lane was quantified by using a scanner (Hewlett-Packard Scan
Jet II cx) and the computer program ImageQuant (Molecular Dynamics) for
autoradiograms; phosphor images were quantified by
using Opti-Quant (Packard). Apparent affinity constants
(Ka)
were calculated for each lipoprotein-biglycan interaction by using SAAM
computer software. Lipoprotein binding to
[35S]SO4-biglycan
was confirmed by staining the gel with oil red O (final concentration
0.3% [wt/vol]).
Statistical Analysis
Significant differences between mean values from
apoE-free and apoE-containing HDL subclasses were determined by ANOVA.
A value of P<0.05 was
considered statistically
significant.
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Results |
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HDL2 and HDL3 were isolated by sequential ultracentrifugation and further separated into apoE-containing and apoE-free fractions with the use of heparin-Sepharose chromatography. HDL2 and HDL3 that did not bind to heparin-Sepharose was found by Western blot analysis to be free of apoB (data not shown) and apoE (Figure 1
). HDL3 that eluted from the
column with 1 mol/L NaCl was found by Western blot to contain apoE
(Figure 1) but not apoB (data not shown). This was confirmed
by immunonephelometry, which was used to quantify apoE, in addition to
apoA-I and apoA-II concentrations. Preliminary experiments showed that
HDL2+E usually contained a small amount of apoB.
Therefore, in subsequent experiments, apoB-containing particles were
removed routinely by using the immunomagnetic precipitation method
described earlier. This procedure was found to specifically remove only
apoB-containing lipoproteins (data not shown).
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HDL2 accounted for 15% to 20% of
the total HDL, and 5% to 6% of each of the HDL subclasses were found
to contain apoE. HDL2+E was characterized as
having significantly higher cholesterol and lower
phospholipid concentration than the other HDL preparations
(Table).
In addition, HDL2+E consistently
contained a 2- to 3-fold greater proportion of protein as apoE compared
with HDL3+E and a 2-to 3-fold lower proportion
of protein as apoA-I compared with HDL3+E and
HDL-E (data not shown).
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Using an electrophoretic gel mobility shift assay, we
demonstrated that apoE was required for the binding of
HDL2 and HDL3 to purified
[35S]SO4-labeled
biglycan. HDL2-E and
HDL3-E did not bind to biglycan
(Figure 2, top panels), demonstrated by the fact that even at
the highest concentration of HDL-E (1.5 mg/mL), there was no
[35S]SO4-biglycan
complexed to the lipoprotein at the origin of the gel. Conversely,
HDL2+E and HDL3+E were
bound to
[35S]SO4-biglycan,
as shown by the increasing radioactivity at the origin and the midpoint
of the gel (bound) and a loss of
[35S]SO4-biglycan
from the front of the gel (free), with increasing concentrations of HDL
(Figure 2, bottom panels). The location of the bands
representing bound
[35S]SO4-biglycan
on the gel is a function of the relative charge of the HDL+E, such that
the more negatively charged the particle, the further it will migrate
into the gel. Thus, a greater proportion of
[35S]SO4-biglycan
bound to HDL2+E and migrated part of the way
into the gel compared with the
[35S]SO4-biglycan
that associated with HDL3+E
(Figure 2, bottom panels), because
HDL2+E was able to bind more biglycan, and the
complex was slightly more negatively charged.
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The extent of binding for HDL2+E and
HDL3+E to biglycan was dependent on the amount
of apoE in the lipoprotein preparation, such that there was greater
binding of biglycan to those HDL preparations that contained more apoE
(data not shown). This suggests that apoE content is a major
determinant of the interaction of HDL+E with biglycan. However, the
apoE content only correlated to the extent of binding when
HDL2+E and HDL3+E were
analyzed separately. This was due to the observation that
HDL2+E was bound significantly greater to
biglycan, even after correcting for the amount of apoE, as shown in the
binding curves for the interaction of
[35S]SO4-biglycan
with HDL2+E and HDL3+E
(Figure 3). Consistent with this, the affinity
constant
(Ka) for
the interaction of
[35S]SO4-biglycan
with HDL2+E was lower than that for the
interaction with HDL3+E
(2.2x10-6 and
7.3x10-6 mol/L, respectively), indicating
that HDL2+E was bound to biglycan with higher
affinity.
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Western blot analysis demonstrated that apoE on
HDL3 was cleaved into 22- and 12-kDa fragments,
whereas apoE on HDL2 remained intact (34 kDa,
Figure 1
). Several inhibitors designed to target
a wide array of proteases were added to plasma and blood during
lipoprotein isolation, but the cleavage of apoE on
HDL3 was observed consistently,
suggesting that this cleavage occurred in vivo. Thus, the lower binding
of HDL3+E to biglycan compared with
HDL2+E may be the result of a loss in the
structural integrity of apoE on HDL3. This was
further supported by the observation that when apoE on
HDL2 was cleaved as a result of long-term
storage
(Figure 4A), the degree of binding to biglycan became similar
to that of HDL3+E
(Figure 4B). Therefore, the presence of the intact 34-kDa
apoE molecule appears to be necessary for optimal biglycan binding, and
the proteolytic cleavage of apoE on HDL3 reduces
its binding to purified arterial wall biglycan compared
with HDL2+E.
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To ensure that the cleavage of apoE did not affect the accuracy of its quantification by immunonephelometry, apoE on HDL was systematically cleaved in the presence of thrombin25 before apoE quantification. Thrombin treatment resulted in a time-dependent increase in the amounts of 22- and 12-kDa cleavage products of apoE, with a corresponding loss in the intact 34-kDa molecule, as detected by Western blot analysis (data not shown). Despite the progressive increase in thrombin-induced cleavage of apoE, the immunonephelometric measurements were essentially identical (1.31, 1.33, and 1.38 µmol/L apoE after 1-, 2-, and 4-hour incubations, respectively, compared with 1.30 µmol/L apoE for HDL not subjected to incubation with thrombin). This indicated that apoE measurements were not affected by proteolytic cleavage, and thus, apo E content could be accurately compared between HDL2+E and HDL3+E.
The interaction of lipoproteins with proteoglycans is
thought to be primarily ionic in nature. Therefore, to assess the role
of positively charged amino acid residues on apoE in the interactions
of HDL+E with biglycan, arginine, and lysine residues were neutralized
by using cyclohexanedione and acetylation, respectively.
Incubation of HDL2+E and
HDL3+E with cyclohexanedione for 15 minutes
resulted in 15% to 20% derivatization of arginine residues,
whereas 35% to 40% arginine residues were derivatized after a 2-hour
incubation period (data not shown). However, the ability of
cyclohexanedione-modified HDL2+E and
HDL3+E to bind to biglycan was impaired to an
even greater extent than the degree of arginine residue modification.
This is shown by the fact that binding of HDL2+E
and HDL3+E to biglycan was reduced 70% and
50%, respectively, after a 15-minute incubation with cyclohexanedione.
Treatment for 2 hours conferred a complete loss of biglycan binding
ability
(Figure 5), even though 60% to 65% of the arginine residues
were not modified. In a similar manner, subjecting
HDL2+E and HDL3+E to
acetylation resulted in an 40% decrease in the positive
charges on lysine (data not shown), which abolished its ability to bind
to biglycan
(Figure 5). Thus, it appears that a limited number of
arginine and lysine residues are functionally important in the binding
of apoE-containing HDL to biglycan. It is most likely that these
functionally important arginine and lysine residues reside in the
heparin binding site(s).
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It has been suggested that an antiatherogenic role of apoE
may result from the ability of apoE to compete with apoB for
proteoglycan binding sites in the artery wall, thus preventing the more
atherogenic apoB-containing lipoproteins from being retained.
Therefore, to investigate how apoE and apoB compare in their binding
affinities for purified extracellular arterial wall
proteoglycans, HDL2+E and LDL were
analyzed for their ability to bind to
[35S]SO4-biglycan
with the use of equimolar concentrations of apoE (for
HDL2+E) or apoB (for LDL). Results indicate that
apoB on LDL was bound to biglycan with a much higher affinity compared
with apoE on HDL2. ApoB on LDL had an 10-fold
lower affinity constant
(Ka) and
thus markedly greater affinity for biglycan compared with apoE on
HDL2 (1.7x10-7
versus 2.2x10-6 mol/L, respectively). These
results suggest that apoE-containing HDL is not likely to be an
efficient competitor with LDL for binding to arterial wall
proteoglycans.
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Discussion |
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In the present study, we have evaluated the role of apoE in the interaction of HDL subclasses with biglycan, a proteoglycan that is 1 of the major extracellular proteoglycans found in human atherosclerotic lesions.6 7 8 We show that HDL2+E binds to biglycan to a significantly greater extent than does HDL3+E, even after correcting for the amount of apoE in these HDL subclasses. Given that all of the HDL preparations (with and without apoE) contain apoA-I and apoA-II, these data indicate that it is the apoE rather than these other apolipoproteins that directly determines the binding of HDL to biglycan. It also suggests that the conformation and/or structure of apoE differs among HDL subclasses and that this may be important in mediating the differences in the binding of biglycan to HDL2+E compared with HDL3+E.
Human apoE contains 299 amino acid residues (34 kDa) and
consists of 2 primary domains separated by a protease-sensitive region.
We show by SDS-PAGE and Western blotting that apoE on
HDL3 is partially cleaved into these 22- and
12-kDa protease-generated fragments, whereas the apoE on
HDL2 is intact (34 kDa). This cleavage cannot be
inhibited with protease inhibitors added immediately after
blood withdrawal, which suggests that apoE may be preferentially
cleaved on HDL3 in vivo. ApoE contains 2
heparin-binding sites, 1 within the 22-kDa (amino terminal) domain and
1 within the carboxyl 12-kDa domain, which apparently is masked in the
presence of lipid.35
Therefore, only the first heparin-binding site of apoE would be
accessible when associated with HDL. Despite the fact that the LDL
receptor and primary heparin-binding sites exist in the carboxyl
domain, previous investigations have reported that apoE on VLDL and
dimyristoyl phosphatidylcholine liposomes must exist as an intact
34-kDa molecule to have optimal receptor and heparin-binding
activities.25 Our present
findings suggest that the presence of an intact apoE molecule on HDL is
also necessary for optimal binding to biglycan. Indeed, preparations of
HDL2 in which the apoE was cleaved into 22- and
12-kDa fragments as a result of long-term storage was shown to have
reduced binding relative to the intact apolipoprotein.
The conformation of apoE has also been shown to be important in its interaction with ß-amyloid, an amyloidogenic peptide that aggregates and becomes the primary component of senile plaques.36 The difference in the binding of apoE3 and apoE4 to ß-amyloid is dependent on the native state of apoE, such that the differential binding was abolished when the apoE was purified (ie, delipidated and denatured). This suggests that the conformation of apoE is affected by its association with other components, such as lipids, and that differences in its conformation can affect its biological activity.
In the present study, selective chemical modifications using cyclohexanedione and acetylation demonstrate that arginine and lysine residues on apoE are important for the interaction of HDL2+E and HDL3+E with biglycan. These findings support the idea that the interaction of apoE with proteoglycans is ionic in nature.4 5 Thus, the interaction would expect to be modulated by alterations in the charge of HDL, such as oxidation or sialic acid content. The composition and size of the HDL particle may also affect the conformation of apoE and thus the accessibility of positive charges on apoE to endogenous proteases and to proteoglycans. Because HDL3 particles are smaller than HDL2 particles, apoE may be more exposed on the surface and thus susceptible to cleavage by proteases.
We also demonstrate in the present study that apoB100-mediated binding of LDL to biglycan is greater than apoE-mediated binding of HDL2 and HDL3. This contrasts with the binding of apoE and apoB100 to the LDL receptor, in which a single molecule of apoE binds with similar affinity as a molecule of apoB100.37 38 Canine apoE HDL cholesterol (HDLc) has been reported to have a higher affinity for the LDL receptor than LDL, which is thought to be due to the ability of multiple apoE molecules on HDLc to bind to multiple LDL receptors, whereas a single apoB molecule on an LDL particle binds 1 LDL receptor molecule.14 37 38 ApoE HDLc is a class of lipoproteins from cholesterol/fat-fed animals that does not contain any other apolipoproteins, which is a different class of HDL than that used in the present investigation. The presence of other apolipoproteins (eg, apoAs and apoCs) most likely affects the interaction of apoE with biglycan by altering the conformation of apoE on HDL. This difference may also be another example of how lipoprotein interactions with the LDL receptor are similar, but not identical, to interactions with proteoglycans. In studies using mice engineered to express wild-type or mutant human apoB, Boren et al39 demonstrated that specific amino acid substitutions in the putative LDL receptor binding region on apoB impaired the binding of LDL to arterial wall proteoglycans but not to the LDL receptor. HDL and apoE participate in reverse cholesterol transport, a process that is believed to protect against the development of atherosclerosis.
The in vivo roles of HDL2 compared with HDL3 regarding cholesterol metabolism and antiatherogenic functions are not well understood, although there are differences between these subclasses. Epidemiological evidence suggests that low levels of HDL2 are more predictive of cardiovascular disease than low levels of HDL3.40 41 Lipid-lowering drugs, hormone replacement therapy, exercise, and dietary interventions all tend to have a greater impact on levels of HDL2 than HDL3.41 In addition, kinetic data suggest that smaller HDL particles within the HDL density range are converted in a unidirectional manner to larger HDL2-sized particles.42 HDL3 is thought to be a greater acceptor of cholesterol, which, on conversion to HDL2, can effectively perform the function of reverse cholesterol transport.42
It may be that apoE has different functions within the artery wall, depending on its cellular association and whether it is produced locally or transported there via circulating lipoproteins. Endogenous expression of apoE by macrophages has been suggested to have more antiatherogenic functions than cell surface proteoglycan-associated apoE.43 44 The presence of apoE within the extracellular matrix also could be potentially atheroprotective. ApoE could compete with apoB for binding sites within the extracellular matrix, which might prevent LDL from accumulating. However, data in the present study suggest that apoE is not likely to be an effective competitor with apoB for biglycan, given the markedly lower binding affinity of apoE on HDL2 compared with apoB on LDL. However, apoE could displace or compete for binding with apoB via other components of the extracellular matrix, an area that deserves further investigation.
On the other hand, apoE-containing HDL may have other roles that are not antiatherogenic and may involve lipid accumulation and smooth muscle cell proliferation and differentiation.15 Furthermore, apoE-containing HDL that is bound to and retained by proteoglycans may be susceptible to oxidation,45 after which it could be taken up by macrophages and be potentially atherogenic.46 Also, apoE-containing HDL that has been retained in the vascular extracellular matrix may not be able to effectively function in reverse cholesterol transport. Thus, the apoE-mediated retention of a subset of HDL by proteoglycans in the artery wall by the mechanisms demonstrated in the present in vitro study may actually promote the development of atherosclerosis.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants HL-30086, DK-02456, DK-07247, HL-07028, and HL-18645. We gratefully acknowledge Christina Tsoi and Thomas F. Johnson for expert technical assistance and Julie Kirk for assistance with preparation of the manuscript.
Received August 23, 2000; accepted October 5, 2000.
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