© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Well-Defined Regions of Apolipoprotein B-100 Undergo Conformational Change During Its Intravascular Metabolism
From the Lipoprotein and Atherosclerosis Research Group and the Departments of Pathology and Biochemistry, Microbiology, and Immunology, University of Ottawa Heart Institute (X.W., J.B., R.W.M.), Ottawa, Ontario, Canada; the Sino-German Laboratory, Cardiovascular Institute (X.W.), Fuwai Hospital, Chinese Academy of Medical Sciences, Beijing, China; and the Department of Biochemistry and Molecular Biology, University College London (R.P.), London, UK.
Correspondence to Ross Milne, PhD, Room H450, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y 4W7. E-mail rmilne{at}ottawaheart.ca
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Abstract |
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Abstract—Apolipoprotein B (apoB)-100–containing lipoproteins are secreted from the liver as large triglyceride-rich very low density lipoproteins (VLDLs) into the circulation, where they are transformed, through the action of lipases and plasma lipid transfer proteins, into smaller, less buoyant, cholesteryl ester–rich low density lipoproteins (LDLs). As a consequence of this intravascular metabolism, apoB-containing lipoproteins are heterogeneous in size, in hydrated density, in surface charge, and in lipid and apolipoprotein composition. To identify specific regions of apoB that may undergo conformational changes during the intravascular transformation of VLDLs into LDLs, we have used a panel of 29 well-characterized anti-apoB monoclonal antibodies to determine whether individual apoB epitopes are differentially expressed in VLDL, intermediate density lipoprotein (IDL), and LDL subfractions isolated from 6 normolipidemic subjects. When analyzed in a solid-phase radioimmunoassay, the expression of most epitopes was remarkably similar in VLDLs, IDLs, and LDLs. Two epitopes that are close to the apoB LDL receptor–binding site show an increased expression in large (1.019 to 1.028 g/mL), medium (1.028 to 1.041 g/mL), and small (1.041 to 1.063 g/mL) LDLs compared with VLDLs and IDLs, and 2 epitopes situated between apoB residues 4342 and 4536 are significantly more immunoreactive in small and medium-sized LDLs compared with VLDLs, IDLs, and large LDLs. Therefore, as VLDL is converted to LDL, conformational changes identified by monoclonal antibodies occur at precise points in the metabolic cascade and are limited to well-defined regions of apoB structure. These conformational changes may correspond to alterations in apoB functional activities.
Key Words: apolipoprotein B • intravascular metabolism • lipoproteins
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Introduction |
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ApoB-100, a 550-kDa glycoprotein composed of 4536 amino acid residues, is a predominant protein component of VLDLs, IDLs, and LDLs1 2 and is a ligand for the LDL receptor (LDLr). It is synthesized in the liver and secreted in the form of large triglyceride-rich VLDLs. Within the circulation, VLDLs are transformed into smaller, denser, cholesteryl ester–rich LDLs through the combined action of plasma lipid transfer proteins, lipoprotein lipase, and hepatic lipase, a process that is accompanied by the loss of all apolipoproteins with the exception of apoB-100. As a consequence of this intravascular remodeling, apoB-containing lipoproteins (LpBs) are constituted of subpopulations of particles that differ in terms of particle diameter, hydrated density, surface charge, and apolipoprotein and lipid composition. This heterogeneity in the physical and chemical properties of LpB leads to apoB conformational heterogeneity that is manifested in the differential accessibility of protease-sensitive sites3 4 and expression of apoB epitopes.5 6 7 This has functional consequences, particularly in terms of the ability of apoB to mediate binding to the LDLr.7 8 The LDL fraction itself is composed of discrete subfractions of particles that differ in their physical and chemical properties, and LDLs are heterogeneous in terms of apoB conformation,9 10 11 12 epitope expression,9 10 11 13 14 accessibility of protease-sensitive sites,15 and affinity for the LDLr.9 10 11 16 17 Whether it is lipid composition, particle diameter, or another variable that is the major modulator of apoB conformation in LDL remains controversial. LDL heterogeneity is thought to have important clinical implications because a predominance of small dense LDL particles has been reported to be a risk factor for atherosclerosis.18
Immunoelectron microscopic analyses suggest that apoB-100
adopts an extended structure on the surface of the LDL and wraps around
the particle.19 20 Unlike most of the other smaller
exchangeable apolipoproteins that are thought to be constituted
primarily of tandem amphipathic 
-helices, apoB-100 is predicted to
contain amphipathic -helices and amphipathic
ß-sheets.1 2 On the basis of a number of experimental
criteria, including the susceptibility of apoB to limited proteolytic
digestion21 and the distribution of lipophilic regions
within apoB primary structure,22 it has been proposed that
apoB-100 is organized into distinct structural domains. A pentapartite
model of apoB domain organization has also been proposed on the basis
of the predicted apoB-100 secondary structure.23 24 A
model of the tertiary structure of the amino-terminal region of apoB
has recently been developed by homology modeling with the use of the
atomic coordinates of lamprey lipovitellin.25 26 27
ApoB-100, lipovitellin, and microsomal triglyceride
transfer protein show homology over a sequence of 
670 amino acids at
the amino termini of their respective primary structures.
In the present study, we describe an immunochemical analysis of apoB in LpBs fractionated as a function of their hydrated density. The present study is unique in that we have used a panel of 29 well-characterized anti-apoB monoclonal antibodies (mAbs) whose corresponding epitopes are distributed throughout the apoB primary structure. We demonstrate that at precise steps during its intravascular metabolism, apoB undergoes important conformational changes that are limited to well-defined regions of its structure.
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Methods |
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Preparation and Characterization of Lipoproteins
Fresh plasma from normolipidemic subjects was prepared from blood obtained from the Canadian Red Cross. The plasma was supplemented with 0.5 mmol/L EDTA, 0.02% NaN3, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 0.5 µg/mL leupeptin. After the removal of chylomicrons, VLDLs (density <1.006), IDLs (density 1.006 to 1.019), and LDLs (density 1.019 to 1.063) were isolated by sequential ultracentrifugation.28 LDL subfractions were isolated by discontinuous density gradient ultracentrifugation of LDLs as described by Teng et al.13 Three fractions (1.019 to 1.028 g/mL [large LDL], 1.028 to 1.040 g/mL [medium LDL], and 1.040 to 1.050 g/mL [small LDL]) were recovered; they were characterized in terms of particle diameter by electrophoresis on a 4% to 15% polyacrylamide gradient under nondenaturing conditions.29 Total and free cholesterol and phospholipid and triglyceride content were determined enzymatically by using kits from Boehringer-Mannheim according to the manufacturer’s recommendations. For LDL, protein concentration was determined by the modified Lowry method (Markwell et al30 ) with BSA used as the standard. To determine apoB protein content of VLDL and IDL, apoB was precipitated with 50% isopropanol, and the protein content of the precipitate was measured after solubilization with 2% sodium deoxycholate.31
Production of New Anti-Human LDL mAbs
New mAbs used in the present study were generated from
mice that had been subjected to a subtractive immunization
protocol32 that will be described in detail elsewhere.
After cyclophosphamide-mediated suppression of the immune response to
normal human LDLs, female BALB/c mice were immunized with LDLs isolated
from a subject with familial defective apoB with use of
N-acetylmuramyl-L-alanyl-D-isoglutamine
(Calbiochem) as an adjuvant. Protocols for cell fusion, screening for
hybridomas by solid-phase immunoassay, and subcloning have been
described in detail.33 None of the mAbs used in the
present study discriminates between normal LDL and familial
defective apoB LDL.
Mapping the Epitopes for Monoclonal Antibodies
The epitopes of the mAbs were mapped within apoB primary
structure by their reactivity with (1) T4 (residues 1 to 1297), T3
(residues 1298 to 3249), and T2 (residues 3250 to 4536) fragments of
apoB generated by thrombin digestion of LDL,34 35 (2) apoB
fragments produced as ß-galactosidase fusion proteins in
Escherichia coli,34 and (3)
carboxy-terminally truncated apoB and apoA-I/apoB fusion proteins
produced by appropriately transfected McA-7777 cell
lines.36 37 In the latter case, mAbs were tested for
reactivity with denatured apoB by Western blotting after SDS-PAGE and
with native LpBs by use of a sandwich
radioimmunoassay.33
LDLr Binding Assay
To assay the reactivity of LDL subclasses with the LDLr,
samples were tested for their ability to compete with
125I-LDL for binding to the LDLr on the surface
of cultured human fibroblasts.38 Methods to determine the
abilities of anti-apoB mAbs to block the binding of LDL to the LDLr and
to bind to LDL in LDL-LDLr complexes have been described
previously.39
Competitive Radioimmunoassay
The competitive apoB radioimmunoassay has been described
previously.40
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Results |
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Production and Mapping the Epitopes of mAbs for ApoB
Although many new anti-human apoB hybridomas were obtained from a total of 8 fusions, we characterized only the mAbs that showed specificity different from those of our previous panel. Antibodies 376, 746, and 3E11 reacted with T4; antibodies 374, 1C4, 3A5, 2G4, 3G9, 278, and 390 reacted with T3; and antibodies 588, 4H11, 3E8, 234, and 6O5 reacted with T2. A summary of the reactivities of all the antibodies with the apoB–ß-galactosidase fusion proteins is presented in Table 1
.
Autoradiograms for 2 of the mAbs that are of particular
importance in the present study are presented in Figure I,
which is published online only (http://atvb.ahajournals.org). The
reactivities of certain mAbs with lipoproteins containing
carboxy-terminally truncated human apoB variants that were expressed in
McA-7777 cells were determined and used to map the corresponding
epitopes more precisely. Antibody 374 reacts with apoB-34 (residues 1
to 1542) but not with apoB-29 (residues 1 to 1306), mAb 1C4 recognizes
apoB-42 (residues 1 to 1880) but not apoB-37 (residues 1 to 1695), and
3A5 reacts with apoB-46 (residues 1 to 2100) but not with apoB-42
(results not shown). A map of the epitopes of the new mAbs, along with
those of the previously described anti-human apoB mAbs that are used in
the present study, is presented in Figure 1.
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The epitopes of several of the mAbs are located within the region
of apoB that includes the LDLr-binding site. The epitopes for mAbs 278,
3G9, and 390 have been assigned to the region of residues 2658 to 3268,
and all 3 mAbs mutually compete with 4G3 (residues 2980 to 3084) but
not with 3F5 (residues 2835 to 2922) for binding to
immobilized LDL (not shown). The 278 epitope differed from
the 4G3, 390, and 3G9 epitopes by being resistant to reductive
methylation (not shown). mAb 588 was mapped to a region between apoB
residues 3687 and 4081. Partial (25%) mutual competition between 5E11
(residues 3441 to 3506)34 and 588 for binding to
immobilized LDL was observed, whereas mAb MB47 (residues
3429 to 3453 and residues 3507 to 3523)41 did not compete
with 588 (results not shown). All mAbs were tested for their ability to
block binding of 125I-LDL to the LDLr on cultured
human fibroblasts. As would be predicted, mAbs specific for epitopes
that are outside the previously defined LDLr-binding region of
apoB39 did not block the binding of LDL to the LDLr
(Figure 2A). Antibodies 278, 3G9, 390,
and 588 did neutralize LDL binding to the LDLr, although in the case of
mAb 390 and 588, neutralization was only partial. The accessibility of
all epitopes in LDL-LDLr complexes was also tested. The 278 and 3G9
epitopes are inaccessible on LDL-LDLr complexes, whereas the epitopes
for mAbs 390 and 588 are partially accessible (Figure 2B).
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Immunoreactivity of LpB
To analyze how apoB conformation changes during the
intravascular metabolism of LpB, we have examined the
expression of apoB epitopes in VLDL, IDL, LDL, and LDL subfractions
isolated from the plasma of 6 normolipidemic individuals. The mean
compositional analysis of the isolated lipoprotein fractions
and LDL subfractions is presented in Table 2. The LDL subfractions were further
analyzed by nondenaturing PAGE. The expected inverse relation
between LDL buoyant density and LDL particle diameter was observed (not
shown). We refer to the 1.019 to 1.028 g/mL, 1.028 to 1.040 g/mL, and
1.040 to 1.063 g/mL density subfractions as large, medium, and small
LDLs, respectively. The large, medium, and small LDL subfractions
contained 6±3%, 68±12%, and 26±8% of the total LDL apoB protein,
respectively. Compared with large and medium LDLs, the small LDLs are
depleted in cholesteryl ester and relatively enriched in
triglycerides. Although there is slightly more apoE
associated with large LDL by SDS-PAGE, no significant differences were
seen between the 3 subfractions in their ability to bind to the LDLr on
cultured human fibroblasts (results not shown). Furthermore, binding
was not influenced by the inclusion of a mAb that blocks apoE-mediated
lipoprotein binding to the LDLr.
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Immunoreactivities of VLDL, IDL, LDL, and LDL subfractions in each
subject were examined by solid-phase competitive radioimmunoassay with
the use of 29 mAbs specific for epitopes that range from the amino
terminus to the carboxy terminus of apoB-100 (Figure 1
).
Representative competition curves for 2 of the mAbs,
1D1 and 4H11, are presented in Figures II and III,
respectively, which are published online only
(http://atvb.ahajournals.org). The immunoreactivities for all the mAbs
are shown in Figure 3. Because the
individual mAbs differ considerably in their respective binding
affinities for LpB, for presentation purposes, we have
normalized the ED50 value to that of LDL of each
subject, to which we have given a value of 1. Most of the epitopes that
we have analyzed were not differentially expressed in the
different lipoprotein fractions. In contrast, 2 epitopes, 4H11 and 6O5,
situated between residues 4342 and 4536, were 3 times more reactive
in LDLs than in VLDLs or in IDLs. Similarly, 2 mAbs, 278 and 4G3, which
mutually compete for binding to immobilized LDLs and whose
epitope(s) is situated in the region of apoB residue 3000, show higher
reactivity with LDLs than with VLDLs or IDLs.
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The large, medium, and small LDL subfractions were similarly
analyzed for differential expression of apoB epitopes, and the
immunoreactivities are presented in Figure 4. Again, to facilitate
presentation of the results, the ED50
values for the subfractions from each subject were calculated relative
to the ED50 value for the medium LDL, which was
assigned a value of 1. Only the epitopes 4H11 and 6O5 were
differentially expressed, with the large LDL being less immunoreactive
than the medium and small LDL.
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Discussion |
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Our results show that at least 2 specific regions of apoB-100 undergo a major conformational change as LpBs are metabolized in the plasma of normolipidemic individuals. In the case of the carboxy terminus of apoB, as defined by the 6O5 and 4H11 epitopes, this change in conformation does not appear to be gradual but occurs abruptly as large buoyant LDLs (density 1.019 to 1.028 g/mL) are converted to particles of density 1.028 to 1.040 g/mL. This conclusion is based on the observation that the 2 epitopes, situated between residues 4342 and 4536, show an
3-fold higher expression in total LDL compared with
VLDL and IDL (Figure 3) and in medium and small LDL subfractions
compared with larger more buoyant LDL (Figure 4). Chen et
al15 have also recently proposed that apoB undergoes a
conformational change at this stage in the intravascular
metabolism of LpB. They have shown that there are major
differences in the accessibility of protease-sensitive sites in
apoB-100 between LpB <1.033 and LpB >1.033. These include
Staphylococcus aureus V8 protease-sensitive sites at
residues 1288 and 3199 and cathepsin D–sensitive sites at residues
2702 and 2666/2669. The concomitant change in the 6O5 and 4H11 epitope
expression and in the accessibility of different protease-sensitive
sites may reflect a global, rather than a local, conformational change
of apoB as large buoyant LDL are converted to smaller denser particles.
If this were the case, it is nevertheless surprising that such an
alteration in the overall apoB conformation was not detected by any of
the other 27 mAbs used in the present study. It has been recently
proposed that (on theoretical grounds alone) apoB must adopt different
conformations in different LDL subspecies but that it may be the
tertiary structure of apoB that is predominantly modulated as a
function of LDL size, with the secondary structure left largely
unchanged.12 In fact, circular dichroic measurements
revealed no significant differences in -helical content among the
LDL subclasses that were analyzed in the present study (X.
Wang, T. Neville, D. Sparks, R. Milne, unpublished data, 1999).
Changes in tertiary structure could potentially include domain shifts
that may not be manifested as changes in the immunoreactivity of
epitopes located within domains but may be detected by markers specific
for interdomain linker regions that could include protease-sensitive
sites. It should be noted that none of the mAbs in the present
panel is specific for epitopes that coincide with the
protease-sensitive sites reported by Chen et al.15 The
region of the carboxy terminus of apoB-100 that includes the 6O5 and
4H11 epitopes has been shown to have a high affinity for
lipid22 and a predicted amphipathic -helical structure
(Figure 1).23 24
In addition to the mAbs that are specific for epitopes near the carboxy
terminus of apoB, mAbs 278 and 4G3 also showed differential reactivity
with VLDL, IDL, and LDL. The major change in the expression of the 278
and 4G3 epitopes would appear to occur as IDLs are converted to LDLs.
From the predicted apoB secondary structure, the 278 and 4G3 epitopes
are located in regions that adopt a predominantly amphipathic ß
structure (Figure 1).23 24 These amphipathic ß
domains of apoB have been proposed to bind irreversibly to
lipid.23 It has been demonstrated that when expressed in
rat hepatoma cells as apoB/apoA-I fusion proteins, the amphipathic ß
sequences are sufficient for recruitment of triglyceride
into VLDL-like particles.36 Furthermore, because neutral
lipid is necessary for the expression of epitopes located in these
regions,42 it would appear that the amphipathic ß
domains are intimately associated with the neutral lipid core of LpB.
Two epitopes that have been mapped to a region (residues 1328 to 1878)
that is also predicted to have an amphipathic ß secondary structure
tended to be more reactive in LDLs than in VLDLs, although this did not
reach statistical significance. Because the amphipathic ß domains
appear to be tightly associated with the neutral lipid core of the
lipoprotein, the change in immunoreactivity of epitopes in these
domains could reflect the alterations in the core that occur during the
intravascular metabolism of VLDL.
Apart from the change in immunoreactivity of the 6O5, 4H11, 4G3, and 278 epitopes, the majority of apoB epitopes are remarkably independent of their lipoprotein environment. Several of the mAbs reported in the present study have been previously used to examine changes in epitope expression in LpB subfractions of moderately hypertriglyceridemic patients. In the previous study, expression of the 5E11 epitope was found to increase progressively in LpB subfractions from VLDL1 (subfractions 100 to 400) through LDL because of an increase in the apparent affinity of the mAb.7 Changes in 5E11 immunoreactivity were not observed in the present study, nor was there differential expression of the overlapping/adjacent epitopes, MB47 and 588. The apparently contradictory results for the 5E11 epitope may reflect differences between LpBs isolated from normal and hypertriglyceridemic individuals. It should also be noted that several of the mAbs (2D8, 3F5, and 4G3) have been previously demonstrated to have reduced reactivity with the very small dense LDLs that characterize subjects with hyperbetalipoproteinemia.13
It is unclear whether the changes in epitope expression reported in the
present study are manifestations of the same changes in apoB
conformation that are responsible for the progressive increase in the
ability of apoB to mediate lipoprotein binding to the LDLr that occurs
as VLDL is converted to LDL.7 8 On the basis of previous
observations39 and the results presented in Figure 2, it would appear that the 4G3 and 278 epitopes are close to
the apoB LDLr-binding site in native LDLs. Thus, conversion of VLDLs to
LDLs could lead to an increased accessibility or an altered
conformation of the region of apoB that includes the LDLr-binding site
and the 4G3 and 278 epitopes. Unlike 4G3 and 278, other epitopes (390,
3G9, 5E11, and MB47) that are also close to the LDLr-binding site are
expressed similarly in VLDLs, IDLs, and the LDL subfractions. The 2
epitopes that show the greatest and most reproducible changes in
expression between the LpB density fractions, 4H11 and 605, are located
near the carboxy terminus of apoB. Both epitopes are accessible in
LDL-LDLr complexes, and neither mAb 4H11 nor mAb 6O5 blocks the binding
of LDL to the LDLr. Furthermore, the change in expression of the 4H11
and 6O5 epitopes occurs as large LDLs are converted to medium LDLs,
whereas we observed no differences between large and medium LDLs in
terms of apoB-mediated binding to the LDLr. Nevertheless, the region of
apoB primary structure that includes the 4H11 and 6O5 epitopes is
thought to be close to the apoB LDLr-binding site in native apoB and
has been proposed to be a negative regulator of apoB-mediated binding
to the LDLr.20 43 44 Recently, Borén et
al44 have shown that VLDLs from apoE-deficient mice that
carry a human apoB-80 transgene can bind to the LDLr with relatively
high affinity, whereas VLDLs from apoE-deficient human apoB-100
transgenic mice bind poorly. A model has been proposed in which lateral
movement of the extreme carboxy terminus of apoB on the surface of the
LpB that occurs during conversion of VLDLs to LDLs would expose the
apoB LDLr-binding site.20 44 The increase in the
immunoreactivity of mAbs 6O5 and 4H11 as large LDLs are converted to
medium LDLs may also result from the same putative apoB conformational
change.
We have previously proposed that the portion of apoB primary structure that is closely associated with the cell surface during binding of apoB to the LDLr is limited to a region bounded by apoB residues 3000 to 4000.39 This is based on the observation that the epitope of mAb 3F5 (residues 2835 to 2922) is totally accessible in LDL-LDLr complexes, whereas that of mAb 4G3 (residues 2980 to 3084) is not. Similarly, whereas the epitope for MB43 (residues 4027 to 4081) is only partially accessible, that of mAb Bsol16 (residues 4154 to 4189) is fully accessible. Seventeen other epitopes situated elsewhere within the apoB primary structure were also fully accessible. Therefore, an interesting observation to emerge from our characterization of the new panel of mAbs is that mAb 588, specific for an epitope situated between apoB residues 3687 to 4081, could partially block the binding of LDL to the LDLr and that its epitope is partially accessible in LDL-LDLr complexes. We further demonstrate partial mutual competition between mAb 588 and mAb 5E11 (residues 3441 to 3506) for binding to immobilized LDL, whereas 588 and MB43 do not complete. Thus, mAb 588 further defines the region of apoB primary structure that is in close contact with the cell surface in LDL-LDLr complexes. It has recently been shown that basic amino acids between apoB residues 3359 to 3369 are necessary for the apoB-LDLr interaction.44
In summary, our immunochemical analysis of LpB density subfractions has shown that in spite of major differences in the physical and chemical properties of the lipoproteins, the apoB conformational changes that can be detected by altered epitope expression are localized to limited well-defined regions within apoB structure. Assuming that the density fractions that were analyzed represent discrete intermediates in the intravascular metabolism of LpB, it would appear that there is a progressive increase in the expression of the 2 epitopes that are close to the apoB LDLr-binding site as VLDLs are converted to LDLs. In addition, a change in apoB conformation occurs as large buoyant LDL are converted to smaller less buoyant particles; this conformational change is detectable by the altered expression of epitopes near the carboxy terminus of apoB. Because this is coincident with altered accessibility of protease-sensitive sites elsewhere in the apoB structure,15 it could represent a major change in the configuration of the apoB polypeptide on the LDL surface.
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Acknowledgments |
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This study was supported by the Medical Research Council of Canada (PG-11471). Dr Pease is the recipient of an Intermediate Research Fellowship from the British Heart Foundation. We wish to thank Drs Roger McLeod and Zemin Yao for providing human apoB-transfected rat hepatoma cell lines, Drs Stephen Young, Linda Curtiss, Joseph Witztum (MB43, MB47), and Jean-Charles Fruchart (B2, B4) for monoclonal antibodies, and Dr Yves Marcel for critically reading the manuscript.
Received September 28, 1999; accepted January 18, 2000.
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