From the Laboratoire GlaxoWellcome, Centre de
Recherche, 25 avenue du Quebec, ZA de Courtaboeuf, 91951 Les Ulis
cedex, France, the ¶ Lipoprotein and Atherosclerosis Group,
University of Ottawa Heart Institute, Ottawa Civic Hospital, Ottawa,
Ontario K1Y 4E9, Canada, and the
Medical Research Council
Molecular Medicine Group, Medical Research Council Clinical Sciences
Center, Imperial College School of Medicine, Du Cane Road,
London W12 0NN, United Kingdom
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ABSTRACT |
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The microsomal triglyceride transfer
protein (MTP) is required for the assembly and secretion of
apoB-containing lipoproteins. To investigate the role of MTP in
lipoprotein assembly, we determined the ability of carboxyl-terminally
truncated forms of apoB to be secreted from cells treated with the MTP
inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and
Grand-Perret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3F cells that produce apoB100 and apoB48,
the inhibitor preferentially blocked apoB100 secretion. When the
inhibitor was tested on McA-RH7777 cells stably transfected with
cDNAs encoding human apoB100, apoB72, apoB53, apoB29, and apoB18,
the secretion of apoB100, apoB72, and apoB53 was preferentially
impaired relative to apoB48 and shorter forms. To delineate the region
between apoB48 and apoB53 that has a high requirement for MTP, we used
puromycin to generate a range of truncated forms of apoB in HepG2
cells. The secretion of apoB53 and longer forms of apoB was markedly
affected by low concentrations of the MTP inhibitor (~ 1 µM), whereas apoB51 and smaller forms of apoB were only
affected at higher concentrations (> 10 µM). The
size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The
MTP inhibitor did not alter the density of the secreted lipoproteins, indicating that each apoB polypeptide requires a minimally defined amount of lipid to attain a secretable conformation. Our results suggest that the folding of the domain between apoB51 and apoB53 has a
high requirement for lipid. This domain is predicted to form
amphipathic Apolipoprotein B (apoB)1
mediates the formation of triacylglycerol-rich lipoproteins in the
liver and intestine. In humans, the liver secretes a large form of 4536 residues called apoB100, whereas the intestine secretes apoB48
corresponding to the 48% amino-terminal part of apoB100 (reviewed in
Ref. 1). The two forms derive from the same gene by the process of
site-specific cytidine deamination of nucleotide 6666 of apoB100 mRNA
(2). This generates a stop codon and defines the carboxyl terminus of
apoB48 (3, 4). Rodent hepatocytes produce both apoB100 and apoB48 (5).
A variety of nonsense or frameshift mutations of the human apoB gene
have been described in familial hypobetalipoproteinemia (6, 7).
Affected individuals typically produce carboxyl-terminally truncated
forms of apoB. The lipid:protein ratio and the size of the lipoprotein
are reduced according to the size of apoB truncated forms, leading to
hypolipidemia. This suggests that the length of apoB determines the
amount of lipid that can assemble into a lipoprotein particle.
The intracellular assembly of apoB with lipid in hepatocytes and
enterocytes absolutely requires a microsomal triglyceride transfer
protein (MTP) complex (8-15). Carboxyl-terminally truncated forms of
apoB longer than apoB23 are only secreted as lipoprotein particles,
whereas smaller forms of apoB are secreted with little or no associated
lipid and do not require MTP to attain a secretion-competent conformation (8-10).
The mechanism of triglyceride-rich lipoprotein assembly is not a simple
size-related lipidation of apoB. ApoB48 has been demonstrated to be
lipidated through two discrete steps in rat liver cells (16, 17). The
second lipidation step of apoB48 is not governed by the size of the
apoB polypeptide but seems to be triggered by short hydrophobic
sequences within apoB48 (18). This could explain why intestinal
chylomicrons are more lipid-rich than hepatic very low density
lipoproteins despite the fact that they contain apoB48 rather than
apoB100. Whether or not the second lipidation step requires MTP
activity is still under debate (19, 20). Partial inhibition of MTP has
been reported to affect the secretion of apoB100 but not of apoB48 from
intestinal Caco-2 cells (21, 22). By contrast, the data of Jamil
et al. (23) indicate that the secretion of apoB48 and
apoB100 from McRH7777 is similarly impaired.
Little is known about amino acid sequences within apoB100 that are
involved in the lipoprotein assembly process. Using computer modeling,
Segrest et al. (24) have suggested that apoB100 is composed
of a pentapartite structure of three Materials--
L-[35S]Methionine
(RedivueTM, 37 TBq/mmol) was purchased from Amersham
Pharmacia Biotech. Culture media, additives and fetal calf serum
were obtained from Life Technologies, Inc. The MTP inhibitor 4'-bromo-3'-methylmetaqualone was synthesized by GlaxoWellcome. Molecular weight markers for electrophoresis were RainbowTM
(Amersham Pharmacia Biotech). All other chemicals were from Sigma.
Cell Culture--
HepG2 and Caco-2 cell lines were obtained from
the American Type Culture Collection. The MhAT3F mouse hepatocyte-like
cell line, derived from mice transgenic for SV40 genes under the
antithrombin III promoter, was provided by B. Antoine (26). McA-RH7777
stably transfected with human apoB truncated variants were maintained in culture as described previously (27, 28). Cells were seeded into
24-well plates (200,000 cells/1.7 cm2) containing basal
Eagle's medium supplemented with penicillin and streptomycin (100 units/ml each) and 10% heat-inactivated fetal calf serum in a
humidified incubator (5% CO2) at 37 °C. All cells were
used after 4 days of culture, except for Caco-2 cells, which were used
after 10 days of culture to allow partial differentiation.
Metabolic Labeling--
To avoid problems of fatty acid
availability (29), all experiments were performed in the presence of
oleic acid (0.5 mM) complexed to albumin prepared as
described previously (25). The MTP inhibitor was dissolved in
Me2SO/ethanol (1:9, v/v) at 4 mM and diluted in
ethanol down to 0.01 mM before the addition to the culture
medium. Cells were incubated for 30 min in methionine-free RPMI 1640 medium before the pulse labeling with
L-[35S]methionine (0.4-2 MBq/well, 15-30
min of pulse). The chase was performed by replacement of the medium
with RPMI 1640 medium. In experiments using mhAT3F or Caco-2 cells, the
chase duration was 2 h. For HepG2 cells, apoB truncated forms were
generated by performing a 150-min chase in the presence of 10 µM cycloheximide and 150 µM of puromycin as
described by Boren et al. (30). Stably transfected
McA-RH7777 cells expressing human truncated apoB forms were labeled for
4 h.
ApoB Immunoprecipitation--
Secreted apoB forms were
quantified by immunoprecipitation from culture medium after the
addition of 1 ml of 60 mM Tris buffer, pH 7, 2 mM ETDA, 1% (v/v) Nonidet P-40, 1 M NaCl, 1 mg/ml bovine serum albumin, 36 µg/ml aprotinin, 1 µg/ml antipain,
50 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride.
Immunoprecipitations were performed using goat anti-human apoB
antiserum (Sigma catalog no. 357-25) after preclearing with
gelatin-agarose beads and protein G-Sepharose beads. The
gelatin-agarose beads were added to remove soluble fibronectin. This
was important, since fibronectin could be easily precipitated and has
an apparent molecular mass of 250 kDa. In some experiments, apoAI was
immunoprecipitated by using sheep polyclonal antibodies (Boehringer
Mannheim catalog no. 726478). All of the samples were analyzed by
denaturing polyacrylamide gel electrophoresis (SDS-PAGE) (31) on a
5-12% acrylamide sigmoid gradient gel under reducing conditions.
After drying the gel, the radioactivity was detected using a
PhosphorImagerTM screen (Molecular Dynamics). All
pulse-chase experiments were reproduced at least three times.
Lipoprotein Isolation by Sequential Flotation
Ultracentrifugation--
In some experiments, lipoproteins were
analyzed by sequential ultracentrifugation. The density was adjusted to
1.040 by the addition of KBr. After 2 h of ultracentrifugation at
120,000 rpm in Beckman TLA 120.2 rotor (480,000 × g),
the top fraction was collected for immunoprecipitation, whereas the
bottom fraction was readjusted to 1.070 before another
ultracentrifugation step. The density was thus successively adjusted to
1.040, 1.070, 1.110, and 1.21, and the four lipoprotein fractions plus
the protein fraction (bottom of 1.21 ultracentrifugation) were analyzed
by immunoprecipitation as before.
Isolation of Lipoproteins from the ER Lumen--
In some
experiments, lipoproteins present in the lumen of the ER were extracted
using NaCO3. HepG2 cells were incubated for 30 min in the
presence of oleic acid (0.5 mM) before the pulse labeling
with L-[35S]methionine (2 MBq/well, 10 min of
pulse). ApoB truncated forms were generated by performing a 10-min
chase in the presence of 10 µM cycloheximide and 150 µM of puromycin as before. Cells were washed and
incubated for 30 min on ice with 200 mM of
NaCO3 and a protease inhibitor mixture
(CompleteTM; Boehringer Mannheim). After 30 min of
ultracentrifugation at 100,000 rpm in Beckman TLA 120.2 rotor
(330,000 × g), the supernatant containing the free
lipoproteins was used either for immunoprecipitation or density
determination as described above.
The Secretion of ApoB48 Is Less Sensitive to MTP Inhibition than
ApoB100--
We have previously shown that the MTP inhibitor
4'-bromo-3'-methylmetaqualone decreases apoB100 secretion by human
hepatic cells either in primary culture or in the HepG2 cell line (25). To determine whether or not the secretion of apoB48 was also affected by an MTP inhibitor, we used cells that naturally produce
simultaneously apoB100 and apoB48 due to partial mRNA editing.
MhAT3F is a well differentiated mouse hepatocyte-like cell line (26,
32) that has a protein secretion profile very similar to primary
hepatocytes. Cells were pretreated for 15 min with the MTP inhibitor
prior to pulse labeling with
L-[35S]methionine for 30 min. After a 120-min
chase, secreted apoB were immunoprecipitated. As shown in Fig.
1, apoB100 secretion by mhAT3F cells was
inhibited by submicromolar concentrations of MTP inhibitor, whereas
apoB48 required higher MTP inhibitor concentrations to be affected. At
a 1.5 µM concentration of the MTP inhibitor, apoB100
secretion was almost abolished (18% of control), whereas apoB48
secretion was marginally affected (71% of control). Albumin secretion
remained insensitive to the compound, thus confirming the lack of
toxicity of the MTP inhibitor.
The Caco-2 cell line derives from a human intestinal tumor and produces
both apoB forms because of partial differentiation (33). Caco-2 cells
were treated and labeled as mhAT3F. Although the ratio of apoB100
versus apoB48 was higher in Caco-2 cells compared with
mhAT3F cells (5 and 0.2, respectively), the same type of selectivity
for apoB100 was observed upon treatment with the MTP inhibitor. At 10 µM, apoB100 secretion was reduced to 21% of control,
whereas apoB48 secretion was similar to control (82%).
Such a selectivity suggests that the requirement for MTP-mediated lipid
assembly is not the same for apoB100 and apoB48 in two cell lines from
different species (mouse or human) and tissues (liver or intestine).
Furthermore, the relative sensitivity of both apoB forms toward MTP
inhibition is not correlated with the proportion of apoB100
versus apoB48 secreted by cells. These results confirm that
the observed selectivity to MTP inhibition is due to different
intrinsic properties of apoB100 and apoB48.
The Secretion of ApoB48 from Transfected McA-RH7777 Cells Is Less
Sensitive to MTP Inhibition Than ApoB53--
To confirm the result
that the MTP inhibitor is more deleterious for the assembly and
secretion of apoB100-containing lipoproteins than for apoB48
lipoproteins, we examined the effect of the MTP inhibitor on the
secretion of a series of carboxyl-terminally truncated forms of human
apoB (apoB18, apoB29, apoB53, apoB72, and apoB100) from stably
transfected rat hepatoma McA-RH7777 cell lines (27, 28). The secretion
experiments were performed in the presence of oleic acid (0.5 mM) to avoid possible lipid deficiency due to apoB
overexpression. Cells transfected with full-length human apoB100
secrete simultaneously apoB100 and apoB48. These cells were compared
with cells expressing human apoB72, apoB53, apoB29, or apoB18. Cells
were labeled with L-[35S]methionine for
4 h in the presence of the MTP inhibitor (10 µM).
This compound dramatically decreased the secretion of apoB100, apoB72,
and apoB53 (less than 20% of control) but had no effect on the
secretion of apoB48, apoB29, and apoB18 (Fig.
2A). As shown in Fig.
2B, the dose response to the MTP inhibitor confirmed that apoB truncated forms can be distinguished in two groups: forms resistant to and forms sensitive to MTP inhibition. The transition between the two groups can be localized roughly between apoB48 and
apoB53.
MTP Inhibition Differently Affects the Secretion of
Carboxyl-terminally Truncated Forms of ApoB--
We have previously
demonstrated the correlation between the concentration of this MTP
inhibitor and the extent of the inhibition of MTP-mediated lipid
transfer activity both in vitro and in cells (25). To
delineate more precisely the relationship between apoB size, MTP
activity, and apoB secretion, we evaluated the ability of a large
series of carboxyl-terminally truncated forms of apoB100 to be secreted
by HepG2 cells. To this end, we used the method described by Boren
et al. (30). This method takes advantage of the capacity of
puromycin to induce premature termination of polypeptide elongation
(34) and allows simultaneous expression of a wide range of apoB
truncated forms. As shown in Fig. 3
(lane a), HepG2 cells pulse-labeled with
L-[35S]methionine for 15 min and chased for
150 min secreted only full-length apoB100. By contrast, if the chase
was performed in the presence of puromycin and cycloheximide, several
apoB truncated forms were secreted (Fig. 3, lane
b). As described by others (30, 35), a relatively
discontinuous pattern of truncated forms was observed. Thus, the
quantification of 26 truncated forms ranging from apoB17 to full-length
apoB100 can be accurately performed (Fig. 3, lane c) and designated as percentage of full-length apoB100 using
human apoB100, apoB72, apoB53, apoB48, apoB29, and apoB18 as molecular mass standards (27, 28).
We next examined the effect of the MTP inhibitor on the secretion of
these 26 truncated forms of apoB. As shown in Fig.
4, low concentrations of the inhibitor
markedly reduced the secretion of apoB polypeptides longer than apoB53.
At higher concentrations, intermediate sized forms of apoB began to be
affected. Smaller truncated forms (apoB17 to apoB26) were insensitive
to high concentrations of the MTP inhibitor.
Importantly, we found that the effect of MTP inhibition was not simply
a linear function of apoB length (Figs. 4 and
5). A clear transition was observed
between apoB51 and apoB53. The concentration of the MTP inhibitor
required to decrease the secretion of apoB51 by 50% (IC50)
was 11.3 µM compared with 1.2 µM for apoB53
(Fig. 5). Even in the absence of oleic acid, the IC50
values for apoB51 and apoB53 still remained different, being 3.2 and
0.46 µM, respectively (data not shown).
These results on HepG2 cells are consistent with our previous findings
using mhAT3F, Caco-2, and transfected McA-RH7777 cell lines and allow
us to define more precisely the critical domain within apoB that
confers a high requirement for MTP activity. This domain is localized
between apoB51 and apoB53, which represents a sequence of less than 100 amino acids.
Inhibition of MTP Does Not Modify the Density of ApoB-containing
Lipoproteins but Decreases the Number of Lipoprotein Particles Secreted
by HepG2 Cells--
Previous studies have established that there is a
direct relationship between apoB size and the density of lipoproteins
secreted from HepG2 cells (30, 35). We studied the effect of the MTP inhibitor on the density of lipoproteins formed with truncated apoB
generated by puromycin treatment. Lipoproteins secreted by HepG2 cells
incubated with or without the MTP inhibitor (1.5 µM) were
analyzed by sequential flotation ultracentrifugation. For each
fraction, apoB was immunoprecipitated and analyzed as before. ApoB
truncated forms smaller than apoB29 were found mainly in the density
>1.21 fraction (Fig. 6, lane
i). This fraction contained also albumin- and lipid-free
proteins (data not shown). ApoB32 to apoB44, apoB46 to apoB63, and
apoB68 to apoB83 were found within densities of 1.21-1.11, 1.11-1.07,
and 1.07-1.04, respectively (lanes g,
e, and c). ApoB100 and apoB90 were mainly found
at density less than 1.04 (lane a). This
correlation between apoB sizes and densities has been described using
various approaches (6, 7, 27, 28, 30, 35-37).
In our hands, inhibition of MTP did not modify the density at which
truncated forms are found, although it decreased the amount of large
truncated forms of apoB (Fig. 6, lanes a-j).
Large forms such as apoB100 or apoB90 were still present at a density
less than 1.040 (Fig. 6, lane b versus
lane a). Similarly, smaller forms such as apoB34
or apoB35 remained in the 1.210-1.110 density range (lane
h versus lane g), and none
of them shifted to the lipid-free fraction in the presence of the MTP
inhibitor (lane j). These results suggest that
the inhibition of the lipid assembly mediated by MTP does not
significantly modify the lipid:protein ratio but rather decreases the
number of secreted particles. Taken together, these results indicate
first that each carboxyl-terminally apoB truncated form requires a
defined amount of lipid to attain a secretable conformation and second
that the inhibition of MTP-mediated assembly with lipid reduces the
number of particles that attain this state. The sequence between apoB51
and apoB53 constitutes a structural domain that is highly dependent on
MTP-mediated lipid transfer for proper folding and secretion.
The Size-related Sensitivity to MTP Inhibition Is Observed in
Nascent Lipoproteins--
Next we examined whether the sensitivity of
truncated forms of apoB longer than apoB53 to MTP inhibition occurred
at the early stages of the lipoprotein assembly process. Following a
10-min chase in the presence of puromycin and cycloheximide, nascent lipoproteins were extracted from the ER of HepG2 cells with
NaCO3. The MTP inhibitor (10 µM)
substantially decreased the number of apoB polypeptides longer than
apoB53 in the lumen of the ER (Fig. 7,
lane b versus lane
a). The sharp transition in behavior between apoB51 and
apoB53 was similar to that observed in our secretion experiment (Figs.
4, 5, and 7, lanes c and d). Likewise,
the densities of the lipoproteins in the lumen of the ER were
comparable with those secreted following a 2-h chase (Fig.
8). These results indicate that the
size-related sensitivity of apoB to MTP inhibition is due to very early
events in the lipoprotein assembly process and not related to
differences in the downstream processing or secretion events. In
addition, we conclude that all of the lipid loaded onto apoB in HepG2
cells takes place within the first 10 min of assembly.
ApoB100 contains a series of structural domains that are predicted
to have different functional roles during the synthesis and assembly of
apoB100 with lipids to form a lipoprotein. The amino terminus of apoB
is rich in disulfide linkages (4, 38-40, 42) and is predicted to have
a compact globular structure that is highly homologous to the ancient
lipid storage and transport protein
lipovitellin.2 The remainder
of apoB forms a belt-like structure wrapped around the surface of the
lipoprotein particle (4, 24, 40, 43-45). The lipid binding structures
of apoB100 are predicted to form two extensive clusters of amphipathic
The structure and the function of the domains of apoB100 can be
assessed by studying the early stages of apoB100 production. Two major
phenomenon seem to be rate-limiting in the production of
apoB100-containing lipoproteins: (i) the translocation of apoB nascent
polypeptide through the ER membrane and (ii) the assembly of
translocated apoB with sufficient lipid to form a secretion-competent lipoprotein (30, 48). Untranslocated apoB remains bound to the ER
membrane and is targeted for degradation (49, 50), although it can be
rescued in certain circumstances (51). It is now clear that the lipid
transfer protein MTP plays an obligatory role in the formation of
apoB-containing lipoproteins (8-11, 19, 52). We have previously shown
that a specific inhibitor of the lipid transfer activity of MTP,
4'-bromo-3'-methylmetaqualone, inhibits apoB100 secretion from hepatic
cells and that this is associated with early presecretory degradation
of apoB (25). Recently, we have shown that apoB degradation can be
observed before the termination of polypeptide elongation in MTP
inhibitor-treated cells, and that this degradation is mediated by the
proteasome (53). This co-translational degradation takes place after
the polypeptide has reached 65% of full length, suggesting that a sequence in this region absolutely requires high MTP activity to escape
the quality control degradation pathway.
To delineate more precisely the domain of apoB requiring
MTP-dependent lipidation, we studied the relationship
between the size of truncated forms of apoB and the efficacy of the MTP
inhibitor to decrease their secretion. First, we found that apoB48
secretion was resistant to the inhibition of MTP compared with
full-length apoB100 both in Caco-2 and mhAT3F cells. Using McA-RH7777
cells transfected with cDNA encoding carboxyl-terminally truncated
forms of human apoB, we showed that two groups of apoB truncated forms could be distinguished. ApoB48 and all smaller forms were not affected
by the MTP inhibitor, whereas the secretion of apoB53 and larger forms
was strongly decreased.
To map more precisely the domain of apoB involved, we have generated a
set of apoB truncated forms ranging from apoB17 to full-length apoB100,
using the property of puromycin to induce premature release of nascent
polypeptides from the ribosomes (34). These apoB truncated forms can be
processed by the cells and secreted as lipoproteins (30, 35). The
density and the particle circumference have been shown to be a function
of the sizes of truncated forms of apoB (35). The same relationship has
been demonstrated using cells transfected with cDNA coding for apoB
truncated forms (27, 28, 35-37). Nevertheless, the role of the MTP in
apoB-containing lipoprotein assembly has never been studied other than
by comparing results obtained in cells that do or do not express MTP.
Here, we progressively reduced MTP activity by increasing
concentrations of a potent and specific MTP inhibitor:
4'-bromo-3'-methylmetaqualone (25). This approach allows the detection
of a discontinuous pattern of sensitivity to MTP inhibition. Truncated
forms smaller than apoB26 were not affected by high concentrations of
the MTP inhibitor. These small forms were found in densities greater
than the 1.21 fraction. This suggests that these forms contain almost no lipid and is consistent with the fact that forms smaller than apoB23
have been reported to be secreted even in MTP-deficient cells (27).
Truncated forms between apoB29 and apoB51 were only affected by high
concentrations (10-40 µM) of the MTP inhibitor, and the
smaller they are the less sensitive they are. Thus, a relatively low
residual MTP activity is sufficient to ensure proper lipidation and
secretion of apoB29 to apoB51 forms. These forms were found at the same
density range as apoAI (data not shown). Surprisingly, a huge gap in
sensitivity to MTP inhibition was observed between forms smaller than
apoB51 and all larger forms. Forms ranging from apoB53 to apoB100 were
very sensitive to submicromolar concentrations of MTP inhibitor,
suggesting that all of them required highly active MTP to be secreted.
The same relationship between size and sensitivity to the MTP inhibitor
was observed for the nascent lipoproteins present in ER lumen after 10 min of chase, indicating that the differential effect of the MTP
inhibitor on lipoprotein secretion occurs during the very early stages
of lipoprotein assembly.
HepG2 cells are deficient in mobilizing lipid stores (54) and produce
apoB100 almost exclusively at a low density lipoprotein rather than a
very low density lipoprotein density (55, 56). Thus, HepG2 cells
represent a model for studying minimally lipidated apoB in which the
second lipidation step responsible for bulk lipid addition is almost
absent. In the present study, we have used this cell system to
establish that MTP inhibition does not modify the density of secreted
apoB-containing lipoproteins, despite decreased production. Thus, a
reduction of MTP-mediated lipid supply has no effect on the lipid to
protein ratio but rather decreases the number of lipoproteins secreted,
as previously suggested (57). This observation indicates that each apoB
polypeptide requires a minimally defined amount of lipid to attain a
secretion-competent conformation.
The size of the domain determining the high sensitivity to the MTP
inhibitor seems very small compared with apoB100 sequence. Experiments
performed on transfected McA-RH7777 cells indicate that this domain is
located between apoB48 and apoB53. Using the puromycin method, we
further mapped this domain to between apoB51 and apoB53, which
encompasses less than 2% of full-length apoB100. The region of apoB
between apoB48 and apoB55 corresponds to an extensive The results we describe in the present paper are consistent with
results published by others and have two major implications for the
understanding of apoB100 lipoprotein assembly. First, they suggest that
special sequences (consecutive amphipathic -helices and to bind lipid reversibly. It proceeds and
is followed by rigid amphipathic
-sheets that are predicted to
associate with lipid irreversibly. We speculate that these domains
enable apoB to switch from a stable lipid-poor conformation in apoB48
to another lipid-rich conformation in apoB100 during lipoprotein assembly.
INTRODUCTION
Top
Abstract
Introduction
References
-helixes alternated with two
-sheets involved in lipid binding. The aim of the present study was
to determine whether the obligatory role of MTP for the assembly and
secretion of very low density lipoproteins is related to a defined
domain of apoB100. To assess this, we compared the secretion from
various cells of a series of carboxyl-terminally truncated forms of
apoB100 in the presence of up to 40 µM of a specific MTP
inhibitor (25). We were able to localize a structural domain within
apoB100 that confers a high requirement for MTP activity, suggesting
that this particular domain plays a key role in the lipoprotein
assembly process.
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
The effect of MTP inhibition on apoB100
and apoB48 secretion by mhAT3F and Caco-2 cell lines. The mouse
hepatocyte-like cell line mhAT3F and human intestinal Caco-2 cell line
were incubated in the presence of oleic acid (0.5 mM) and
labeled with L-[35S]methionine for 30 min
followed by 120 min of chase. The MTP inhibitor was added at various
concentrations 15 min before the pulse. ApoB was immunoprecipitated
before SDS-PAGE, whereas albumin was directly analyzed by SDS-PAGE.
Radioactivity was determined by PhosphorImagerTM screen
autoradiography.
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Fig. 2.
Effect of the MTP inhibitor on the
secretion of human apoB variants by transfected McA-RH7777 cells.
McA-RH7777 cells stably transfected with cDNAs encoding
carboxyl-terminally truncated forms of human apoB were labeled with
L-[35S]methionine for 4 h in the
presence of 0.5 mM oleic acid. The MTP inhibitor was added
15 min before labeling. Secreted apoB was immunoprecipitated before
SDS-PAGE, and radioactivity was determined by
PhosphorImagerTM screen autoradiography. A,
phosphor screen autoradiography of secreted apoB variants in the
presence or absence of 10 µM MTP inhibitor. B,
quantification by scanning of apoB variants secreted in the presence of
various concentrations of the MTP inhibitor.
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Fig. 3.
Puromycin induces the secretion of 26-well
quantifiable apoB truncated forms. HepG2 cells were labeled with
L-[35S]methionine for 15 min and chased for
210 min in the presence (lane b) or absence
(lane a) of 150 µM puromycin and 10 µM cycloheximide. Oleic acid (0.5 mM) was
added 15 min before the pulse. Secreted apoB was immunoprecipitated and
size-fractionated by SDS-PAGE. The radioactivity in apoB
(lane c) was quantified by
PhosphorImagerTM screen autoradiography. The sizes of
truncated forms were determined using human apoB100, apoB72, apoB53,
apoB48, apoB29, and apoB18 (boldface letters)
secreted by transfected McA-RH7777 cells and expressed as percentage of
full-length apoB100.
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Fig. 4.
The effect of increasing concentrations of
MTP inhibitor on the secretion of 26 truncated forms of apoB.
HepG2 cells were pulse-chase-labeled in the presence of 150 µM of puromycin and 10 µM of cycloheximide,
and secreted apoB was immunoprecipitated as in Fig. 3, lane
b. The MTP inhibitor (0.5-40 µM) was added 15 min before the pulse.
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Fig. 5.
The concentration of MTP inhibitor required
to decrease by 50% the secretion of full-length and
carboxyl-terminally truncated forms of apoB. Incubation and
analysis were performed as in Fig. 4. The concentration required to
decrease by 50% the secretion (IC50) of each apoB form was
calculated and plotted as a function of apoB size. The results are the
mean of three experimental observations.
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Fig. 6.
The effect of MTP inhibition on the density
of lipoproteins assembled around truncated forms of apoB. HepG2
cells were labeled in the presence of 150 µM puromycin
and 10 µM of cycloheximide as in Fig. 4. The MTP
inhibitor (1.5 µM) was added 15 min before the pulse.
Secreted proteins were separated by sequential flotation
ultracentrifugation. Density was successively adjusted to 1.040, 1.070, 1.110, and 1.21 with KBr. Fractions corresponding to density less than
1.04 (lanes a and b), between 1.04 and
1.07 (lanes c and d), between 1.07 and
1.11 (lanes e and f), between 1.11 and
1.21 (lanes g and h), or above 1.21 (lanes i and j) were analyzed by
immunoprecipitation. ApoB secretion in control condition
(lanes a, c, e,
g, and i) was compared with apoB secretion in the
presence of a 1.5 µM concentration of the MTP inhibitor
(lanes b, d, f,
h, and j).
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Fig. 7.
The effect of MTP inhibition on truncated
forms of apoB present in the ER lumen. HepG2 cells were treated
and pulse-labeled as in Fig. 4 but with a 10-min pulse. The duration of
the chase period in the presence of puromycin and cycloheximide was 10 min for intracellular lipoproteins (lanes a and
b) that were released from the ER lumen using
NaCO3 (see "Experimental Procedures"). The duration of
the chase period was 120 min for secreted lipoproteins
(lanes c and d). All samples were
analyzed after apoB immunoprecipitation as in Fig. 4. Control cells
(lanes a and c) were compared with
cells treated with a 10 µM concentration of the MTP
inhibitor added 15 min before the pulse (lanes b
and d). Six truncated forms of apoB with estimated sizes of
apoB26, apoB41, apoB50, apoB53, apoB77, and apoB100 are indicated with
dotted arrows (see Fig. 8).
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Fig. 8.
Comparison between the density of
lipoproteins assembled around truncated forms of apoB present in the ER
or secreted. HepG2 cells were treated, pulse-labeled, and chased,
and lipoproteins were extracted as described in the legend to Fig. 7.
Lipoprotein fractions were obtained by sequential flotation
ultracentrifugation as described in the legend to Fig. 6 and analyzed
by immunoprecipitation. The data for six representative apoBs (see Fig.
7) are shown. The densities of NaCO3-extracted lipoproteins
from the ER, after a 10-min chase (closed
symbols) were compared with the densities of secreted
lipoproteins after a 120-min chase (open
symbols). Control cells (circles,
dashed lines) were compared with cells treated
with a 2.5 µM concentration of the MTP inhibitor
(triangles, solid lines). The
densities of fractions A, B, C, D, and E are less than 1.04, between
1.04 and 1.07, between 1.07 and 1.11, between 1.11 and 1.21, and above
1.21, respectively. Results were expressed as percentage of the total
amount of each truncated form found in all fractions at 10 min of chase
(ER lumen) in control cells.
DISCUSSION
-helices, which are predicted to be flexible and bind lipid
reversibly, and two extensive
-sheets, which are predicted to be
rigid and associate with lipid irreversibly (24). The portions of
apoB100 that form the amphipathic
-helical domains are not
susceptible to trypsin digestion (46, 47), indicating that they either
are firmly anchored to the core of the lipoprotein particle or that
they possess a compact globular structure.
-helical
domain (4, 24, 40). Computer predictions and lipid and monoclonal
antibody (41) binding studies suggest that this region is flexible and
that it binds lipid reversibly. Upstream and downstream of this domain
are the two extensive amphipathic
-sheets, which are predicted to
bind lipid irreversibly and to have a much more rigid structure. The
precise role of these domains remains to be defined, but we can
speculate that lipidation by MTP could induce conformational changes in
nascent apoB that are absolutely required for further lipidation and
for lipoprotein assembly. The localization of the
-helical domain
immediately after the end of apoB48 suggests that this domain, possibly
in conjunction with the succeeding
-sheets, allows apoB to switch from one stable conformation (lipid-poor apoB48) to another stable conformation (lipid-rich apoB100) during polypeptide elongation. The
presence in apoB100 of the
-helical domain and
-sheet beyond the
carboxyl terminus of apoB48 must presumably confer on apoB100 the high
level of MTP-mediated lipid transfer activity needed for the
lipoprotein assembly process. The fact that only large apoB nascent
polypeptides undergo co-translational degradation by the proteasome
(53) is consistent with the misfolding of apoB sequences downstream
apoB53 when MTP is inhibited. An alternative explanation for the
inverse relationship between apoB size and sensitivity to MTP
inhibition would be that mild inhibition of MTP reduces the amount of
lipid associated with all forms of apoB at the point they are released
from the ribosome, and if so that underlipidated forms of large
apoB-containing lipoproteins are more susceptible to proteolysis as
they proceed along the secretory pathway than their smaller apoB
polypeptides. The observation that the same relationship exists between
apoB size and sensitivity to the MTP inhibitor for both nascent
lipoproteins, in the ER, and secreted lipoproteins rules out this
possibility and places the effects we observe proximate to
co-translational lipoprotein assembly.
-helical and
-sheet
domains) in apoB100 trigger the lipidation mediated by MTP, which is
required for proper folding. When lipid addition at the site of apoB
assembly is reduced through MTP inhibition, apoB is diverted to a
degradation pathway. Second, they indicate that during early steps of
lipoprotein assembly, the lipid:protein ratio is only determined by the
length of apoB.
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ACKNOWLEDGEMENTS |
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We thank Jorge Kirilovsky for critical reading of the manuscript and Bénédicte Antoine for providing mhAT3F cells.
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FOOTNOTES |
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* 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: Novartis Pharma AG, K-125.10.040, Basel, Switzerland.
** To whom correspondence should be addressed: Laboratoire Glaxo-Wellcome, Centre de Recherche, 25 avenue du Quebec, ZA de Courtaboeuf, 91951 Les Ulis cedex, France. Tel: 169-29-6000; Fax: 169-07-4892; E-mail: tgp28876{at}GlaxoWellcome.co.uk.
The abbreviations used are: apo, apolipoprotein; MTP, microsomal triglyceride transfer protein; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis.
2 Mann, C. J., Anderson, T. A., Read, J., Chester, S. A., Harrison, G. B., Kochl, S., Ritchie, P. J., Bradbury, P., Amey, J., Vanloo, B., Rosseneu, M., Infante, R., Hancock, J. M., Levitt, D. G., Banaszak, L. J., Scott, J., and Shoulders, C. C. (1999) J. Mol. Biol. 285, 391-408.
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REFERENCES |
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