Well-Defined Regions of Apolipoprotein B-100 Undergo
Conformational Change During Its
Intravascular Metabolism
Xingyu Wang, Richard Pease, Jesse Bertinato, Ross W. Milne
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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. (Arterioscler Thromb Vasc Biol. 2000;20:1301-1308.)
Key Words: apolipoprotein B 䡲 intravascular metabolism 䡲 lipoproteins
A
poB-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.5–7
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 –12
epitope expression,9 –11,13,14 accessibility of protease-sensitive
sites,15 and affinity for the LDLr.9 –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
Received September 28, 1999; revision accepted January 18, 2000.
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
[email protected]
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1301
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Arterioscler Thromb Vasc Biol.
May 2000
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␣-helices, apoB-100 is predicted to contain amphipathic
␣-helices and amphipathic ␤-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.25–27 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.
Methods
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 ␮g/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 ␤-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
Results
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–␤-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.
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
Wang et al
TABLE 1.
ApoB-100 Conformational Changes
1303
Immunoreactivity of mAbs With ApoB–␤-Galactosidase Fusion Proteins Expressed in E coli
T4 Fusion
Proteins
97–
474
97–
689
582–
851
851–
1084
995–
1328
851–
1082
995–
1328
Epitope
Position
376
⫺
⫹
⫺
⫺
⫺
⫺
⫺
474–582
746
⫺
⫹⫹
⫺
⫺
⫺
⫺
⫺
474–582
3E11
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
1082–1328
1D1*
⫺
⫹⫹
⫺
⫺
⫺
⫺
⫺
474–539
T3 Fusion
Proteins
1281–
1328
1281–
1480
1281–
1880
1480–
1880
1480–
2240
1693–
2148
2148–
2375
2240–
2658
2658–
3268
2488–
3215
3132–
3494
Epitope
Position
374
⫺
⫺
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
1306–1542†
1C4
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
1696–1880†
3A5
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
1880–2100†
2G4
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹
⫺
⫺
⫺
⫺
2148–2375
3G9
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
⫺
⫺
2658–3268
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278
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
⫺
2658–3268
390
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
2658–3268
4G3*
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹
⫹
2980–3084
T2 Fusion
Proteins
3132–
3494
3214–
3635
3214–
3727
3286–
3452
3351–
3506
3506–
3687
3506–
4081
4081–
4342
4081–
4536
Epitope
Position
588
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹
⫺
⫺
3687–4081
4H11
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
4342–4536
3E8
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹
4342–4536
234
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
4342–4536
605
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
4342–4536
Bsol7*
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
4521–4536
*mAbs 1D1, 4G3, and Bsol7, whose epitopes have been mapped previously,34 were used as controls for epitopes in T4, T3, and T2, respectively.
†See text for additional information used in the mapping of these epitopes.
⫹⫹⫹, ⫹⫹, ⫹, ⫺ indicate strong, moderate, weak, or no reactivity, respectively, of mAb with apoB-␤-galactosidase fusion protein.
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 LDLrbinding 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
Figure 1. Map of apoB
epitopes recognized by mAbs
that have been used in the
present study. A, The primary
sequence of apoB is represented as a thick solid line.
Numbers under the apoB primary sequence are the amino
acid residues that define
regions containing epitopes,
and those above the line are
the epitopes of the mAbs. The
epitopes of mAbs identified in
italics have not been reported
previously. The epitopes of
previously characterized
mAbs34 are shown in normal
font. B, Pentapartite model of
apoB structure.23,24
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Figure 2. Antibody-mediated inhibition of binding of LDL to the
LDLr and the binding of anti-apoB mAbs to receptor-bound
LDL. A, Newly generated mAbs were tested for their ability to
inhibit the binding of 125I-LDL to the LDLr on cultured human
fibroblasts. Several mAbs that were previously shown39 to block
(4G3, 5E11, and 3A10) or not block (1D1) binding of LDL to the
LDLr were included in the assays. Results are presented as the
125
I-LDL bound in the presence of mAb as a percentage of LDL
bound in the absence of mAb. B, 125I-mAbs were also tested for
their ability to bind to LDL that was bound to the LDLr of cultured human fibroblasts. Details for the calculation of the stoichiometry of binding have been described previously.39 All
results are normalized to 2D8, whose epitope has been shown
to be fully exposed in LDL-LDLr complexes. Several other control mAbs whose epitopes were previously shown to be either
exposed (1D1) or inaccessible (4G3 and 5E11) in LDL-LDLr
complexes were also included in the assays.39
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).
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
TABLE 2.
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.
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.
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.
Mass Percentage Composition of VLDL, IDL, LDL, and LDL Subfractions
TG
FC
CE
PL
Protein
VLDL
52.0⫾12.1
8.9⫾1.1
10.9⫾1.5
20.1⫾3.4
8.0⫾0.8
2.8
IDL
23.3⫾1.5
9.4⫾1.5
24.6⫾1.6
21.9⫾0.9
20.9⫾1.5
11.6
LDL
7.8⫾0.7
10.6⫾2.2
38.0⫾4.5
21.3⫾1.1
22.3⫾???
22.3
Large LDL
4.3⫾1.9
9.8⫾2.7
42.9⫾17.3
23.4⫾3.3
19.6⫾3.4
ND
Medium LDL
6.2⫾0.6
11.0⫾2.0
39.9⫾8.0
20.7⫾1.1
22.2⫾2.1
ND
Small LDL
9.2⫾0.8
9.2⫾3.1
39.1⫾3.1
16.9⫾8.7
25.6⫾1.4
ND
Values are mean⫾SEM. TG indicates triglycerides; FC, free cholesterol; CE, cholesteryl ester; and PL, phospholipid.
ApoB
Wang et al
ApoB-100 Conformational Changes
1305
Figure 3. Reactivities of 29 anti-apoB
mAbs with VLDL, IDL, and LDL. A, Immunoreactivity of VLDL, IDL, and LDL from 6
normolipidemic subjects determined in a
solid-phase immunoassay with a panel of
29 anti-apoB mAbs. Results are
expressed as relative ED50, the concentration of competitor required to reduce
binding to 50% of that occurring in the
absence of competitor. The ED50 value
obtained with LDL was normalized to
unity. A paired t test was used to determine the significance of differences
observed in the immunoreactivity
between lipoprotein classes. For mAbs
605, 4H11, 4G3, and 278, there are significant differences in immunoreactivity
between VLDL or IDL and LDL (for mAb
605, P⬍0.001 for VLDL vs LDL, P⬍0.0005 for VLDL vs LDL, and P⫽NS for VLDL vs IDL; for mAb 4H11, P⬍0.01 for VLDL vs LDL,
P⬍0.005 for IDL vs LDL, and P⫽NS for VLDL vs IDL; for mAb 4G3, P⬍0.01 for VLDL vs LDL, P⬍0.01 for VLDL vs LDL, and P⫽NS for
VLDL vs IDL; and for mAb 278, P⬍0.05 for VLDL vs LDL, P⬍0.05 for IDL vs LDL, and P⫽NS for VLDL vs IDL). B, Distribution of
epitopes within domains defined by the pentapartite model of apoB structure.23,24
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Discussion
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 proteasesensitive 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.
Figure 4. Reactivities of 29 anti-apoB mAbs
with LDL density subfractions. A, Immunoreactivity of large, medium, and small LDLs
from 6 normolipidemic subjects. The ED50
value obtained with medium LDL was normalized to unity. The mAbs 605 and 4H11
showed increased reactivity with small and
medium LDLs compared with large LDLs
(P⬍0.001). No significant differences were
found between LDL subclasses for any of
the other mAbs. B, Distribution of epitopes
within domains defined by the pentapartite
model of apoB structure.23,24
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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 ␤ structure (Figure 1).23,24 These amphipathic ␤ 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 ␤ 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 ␤ 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 ␤ secondary structure
tended to be more reactive in LDLs than in VLDLs, although
this did not reach statistical significance. Because the amphipathic ␤ 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 apoB100 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
Wang et al
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.
Acknowledgments
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.
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Well-Defined Regions of Apolipoprotein B-100 Undergo Conformational Change During
Its Intravascular Metabolism
Xingyu Wang, Richard Pease, Jesse Bertinato and Ross W. Milne
Arterioscler Thromb Vasc Biol. 2000;20:1301-1308
doi: 10.1161/01.ATV.20.5.1301
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