(Received for publication, August 31, 1995; and in revised form, January 23, 1996)
From the
Microsomal triglyceride (TG) transfer protein (MTP) is an
endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit
and protein disulfide isomerase. It is believed that MTP delivers TG to
nascent apoB molecules during the assembly of lipoprotein particles in
the secretory pathway. Although in vitro studies have
established the mechanism of TG transfer between donor and acceptor
membranes, the mechanism of action of MTP in vivo remains
unknown. The present studies were undertaken to examine whether or not
the transfer of TG to nascent apoB in the endoplasmic reticulum
involves the physical interaction between MTP and apoB. HepG2 cells
were labeled with [H]leucine, lysed in a
nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP
antiserum. We found that labeled apoB and protein disulfide isomerase
were co-immunoprecipitated by this procedure. In addition, we were able
to detect the 97-kDa subunit of MTP in these immunoprecipitates by
immunoblot. The association of MTP and apoB, as assessed in
pulse-labeled cells by co-immunoprecipitation, was transient; apoB was
prominent on fluorgraphy at 10 min of chase but minimal thereafter.
Oleic acid treatment, which protects apoB from rapid intracellular
degradation by increasing TG availability, increased both the degree
and the duration of association between MTP and apoB dramatically.
Inhibition of TG synthesis by Triacsin D, on the other hand,
significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease
inhibitor, which directly protects apoB from rapid intracellular
degradation but does not affect TG synthesis, increased the interaction
between MTP and apoB only slightly, although it did prolong it. Our
results suggest that direct interaction between MTP and apoB occurs
during the assembly of apoB-containing lipoproteins in HepG2 cells.
The increased secretion rate of apoB-containing lipoprotein
particles from liver results in elevated plasma levels of low density
lipoproteins(1, 2, 3, 4) , a major
risk factor for the development of atherosclerotic diseases. Studies
have demonstrated that apoB secretion is regulated
posttranslationally(5, 6, 7, 8, 9, 10, 11, 12) .
Among factors believed to affect the secretion of apoB-containing
lipoproteins from HepG2 cells, the availability of newly synthesized
triglyceride (TG) ()may be the most
important(13, 14, 15, 16, 17) .
Thus, when lipid availability is increased, rapid intracellular
degradation of newly synthesized apoB is inhibited; inhibition of lipid
synthesis, on the other hand, results in enhanced degradation of apoB.
Despite the large body of evidence demonstrating the importance of hepatic lipids in the assembly and secretion of apoB-containing lipoproteins, TG synthesis appears to be normal, and lipid droplets accumulate, in livers of patients with abetalipoproteinemia(18) , a condition characterized by the absence of apoB secretion. Recently, defective assembly and secretion of apoB-containing lipoproteins in affected patients was found to be associated with mutations in the gene encoding a 97-kDa protein, microsomal TG transfer protein (MTP) large subunit(19, 20) . In vitro studies (21, 22) showed that MTP efficiently catalyzes the transfer of TG and other lipids from donor membranes to acceptor membranes. MTP large subunit forms a heterodimer with protein disulfide isomerase in the lumen of the endoplasmic reticulum (ER) in hepatocytes and enterocytes. MTP large subunit appears to be expressed normally only in hepatocytes and enterocytes; protein disulfide isomerase is ubiquitously expressed. Two recent studies (23, 24) convincingly demonstrated that coordinate expression of large apoB truncations and MTP large subunit in cells that normally do not express either of the two proteins resulted in the efficient secretion of apoB-containing lipoproteins. ApoB was not secreted from these cells before MTP large subunit was expressed.
Although the above studies have clearly indicated a role for MTP in the assembly and secretion of apoB-containing lipoproteins from hepatocytes, the in vivo mechanism underlying this activity remains unknown. An unanswered question is how MTP transfers TG molecules to apoB during the assembly of lipoprotein particles. More specifically, does a physical interaction between MTP and apoB play a role in this process? The present studies were conducted to answer this question.
HepG2 cells were labeled for 4 h with
[H]leucine and lysed in a nondenaturing buffer.
Immunoprecipitation was carried out with anti-human apoB, anti-MTP, or
anti-MTP large subunit antisera under nondenaturing conditions. With
anti-apoB antiserum, apoB100 was precipitated from cell lysates (Fig. 1). ApoB, however, was also precipitated by either
anti-MTP or anti-MTP large subunit antibodies (Fig. 1). The
latter results suggested interaction between apoB and MTP. Anti-MTP
antibody appeared to precipitate a small proportion of the labeled apoB
pool. This is consistent with the finding that only 5-10% of
newly synthesized apoB is secreted from HepG2 cells under basal
conditions(14, 15, 16, 17) .
Figure 1:
Anti-MTP antibodies co-precipitate
newly synthesized apoB in HepG2 cells. HepG2 cells were labeled with
[H]leucine for 4 h, lysed with a nondenaturing
buffer, and immunoprecipitated with anti-apoB, anti-MTP, or anti-MTP
large subunit antiserum. With anti-apoB antiserum, labeled apoB was
precipitated (
-apoB); with anti-MTP large subunit, labeled apoB
was also precipitated (
-MTPL); with anti-MTP, both
labeled apoB and labeled MTP small subunit (protein disulfide
isomerase, 55 kDa) were precipitated (
-MTP).
Since other proteins (with the exception of protein disulfide isomerase; see below) in the cell lysate were not precipitated by the anti-MTP antibodies, it appeared that the MTP-apoB interaction was specific. We were concerned at first that neither the apoB antiserum nor the two anti-MTP antibodies precipitated MTP large subunit (Fig. 1). This is not surprising, however, when one considers that the MTP large subunit has a half-life reported to be more than 100 h(26) . Thus, our protocol would radiolabel a very small proportion of the cellular MTP large subunit pool. On the other hand, the anti-MTP antibody did immunoprecipitate the small subunit of MTP (protein disulfide isomerase) together with apoB (Fig. 1).
To confirm the physical interaction between apoB and MTP, immunoblotting experiments were carried out. HepG2 cells were lysed with the nondenaturing buffer, and immunoprecipitation was carried out with either nonimmune serum or MTP large subunit antiserum. The immunoprecipitates and an aliquot of HepG2 cell whole lysate were run on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blotted with anti-apoB antibodies. As shown in Fig. 2A, apoB was not detected with nonimmune serum; apoB was detected, however, with anti-MTP large subunit antiserum. In another experiment, MTP large subunit was detected in an anti-MTP large subunit immunoprecipitate by immunoblotting with anti-MTP large subunit antiserum (Fig. 2B).
Figure 2:
MTP is associated with apoB in HepG2
cells. HepG2 cells were lysed with a nondenaturing buffer and
immunoprecipitated with nonimmune serum or anti-MTP large subunit
antiserum (-MTPL). The immunoprecipitates and an aliquot
of HepG2 cell whole lysate were run on SDS-PAGE and transferred to a
nitrocellulose membrane. The membrane was blotted with either anti-apoB
antibody (panel A) or anti-MTPL (panel B). In panel A, immunoblotting with anti-apoB antibody demonstrated
that apoB was immunoprecipitated by anti-MTPL antibody. In panel
B, immunoblotting with anti-MTPL demonstrated that MTPL was
co-immunoprecipitated in the same experiment. In a parallel experiment,
HepG2 cell lysates were immunoprecipitated with either nonimmune serum
or anti-human apoB antiserum (
-apoB). The
immunoprecipitates were run on SDS-PAGE and transferred to a
nitrocellular membrane. The membrane was blotted with anti-MTP large
subunit (panel C). The result indicated that MTP large subunit
was co-immunoprecipitated by anti-apoB antibody. The dark band at the bottom is rabbit anti-human
IgG.
HepG2 cell lysates were also immunoprecipitated with nonimmune serum or anti-human apoB antiserum. The immunoprecipitate was run on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blotted with anti-MTP large subunit antiserum. MTP large subunit (97 kDa) was detected in the anti-apoB immunoprecipitate but not in the nonimmune precipitate (Fig. 2C).
Figure 3:
The
MTP-apoB interaction is coordinated with the synthesis of TG. HepG2
cells were preincubated with one of the following agents for 1 h: 1)
1.5% BSA alone, 2) 1.5% BSA plus ALLN (40 µg/ml), 3) 1.5% BSA plus
OA (0.2 mM), 4) 1.5% BSA plus OA (0.2 mM) and TGI
(12.5 µM), or 5) 1.5% BSA plus TGI (12.5 µM).
Cells were then labeled with [H]leucine for 2 h,
lysed with a nondenaturing buffer, and immunoprecipitated with either
anti-apoB or anti-MTP large subunit (panels A and B). Panel B is presented as mean ± S.D. of triplicate
wells. The results demonstrated that treatment with OA (which
stimulates TG synthesis as shown in panel C) increased the
association between apoB and MTP. Co-incubation of OA with TGI
abolished the effect of OA. In parallel experiments, HepG2 cells were
incubated for 2 h with or without [
H]glycerol and
one of the agents described above. For TG determination,
[
H]glycerol-labeled cells were extracted with
hexane/isopropyl alcohol (3/2), and TG synthesis was determined by thin
layer chromatography. Results in panel C are presented as mean
± S.D. of triplicate wells. Changes in TG synthesis paralleled
changes in apoB-MTP association. In a third experiment, unlabeled cells
were directly lysed and immunoprecipitated with anti-MTP large subunit
antiserum. The immunoprecipitates were run on SDS-PAGE, transferred to
a nitrocellulose membrane, and blotted with anti-MTP large subunit (panel D). None of the treatments significantly affected MTP
levels in HepG2 cells.
The results of these two experiments indicated that the MTP-apoB interaction was closely coupled with, or dependent upon, TG synthesis. These results are consistent with a pathway in which MTP is abundant but only binds to apoB in the presence of newly synthesized triglyceride. Additionally, our findings support the idea that a MTP-apoB physical interaction may be important in both the TG transfer process and lipoprotein assembly. On the other hand, the accumulation of apoB, in the absence of increased TG availability (as would occur in the presence of ALLN), does not appear to significantly modulate the MTP-apoB interaction.
Figure 4:
MTP-apoB interaction occurs at an early
stage of the assembly of apoB-containing lipoproteins. HepG2 cells were
labeled with [H]leucine for 10 min and chased in
serum-free medium up to 180 min. At each time point, cells were lysed
and immunoprecipitated with either anti-apoB (
-apoB) or
anti-MTP large subunit (
-MTPL). The highest band on the top of the gel represents aggregated material.
Interaction between MTP and apoB was greatest at 10 min of chase and
rapidly diminished thereafter.
Figure 5:
MTP-apoB interaction parallels the extent
of apoB translocation across ER membranes. HepG2 cells were
preincubated with BSA, OA, or ALLN for 1 h, pulse-labeled with
[H]leucine for 10 min, and chased in serum-free
medium up to 60 min. At each time point of the chase, cells were lysed
and immunoprecipitated with either anti-apoB or anti-MTP large subunit (panel A). OA treatment markedly increased and also prolonged
the association of MTP with apoB. ALLN treatment only increased the
association slightly but prolonged it significantly. Similar results
were obtained in three experiments (panels B and C).
OA was added to some of the ALLN-treated cells after they had been
chased for 30 min, and the cells were chased for an additional
10-30 min in serum-free medium. At each time point, cells were
lysed and immunoprecipitated with either anti-apoB or anti-MTP large
subunit antiserum (panel B). The highest band represents the aggregated material on the top of the gel.
The addition of OA after a 30-min chase, which would increase
translocation of nascent apoB(15) , sharply increased MTP-apoB
binding (compare ALLN at the 60-min time point with ALLN at 30 min
+ OA at the 20- or 30-min time point). Overall, the results
suggest that increased translocation of apoB into the ER lumen (OA
treatment) is associated with greater interaction of apoB with MTP. On
the other hand, simply increasing apoB content without effective
translocation (ALLN treatment) mainly prolongs the low, basal level of
interaction.
In summary, the present studies demonstrate that the physical interaction between MTP and nascent apoB participates in the assembly of apoB-containing lipoproteins. This interaction is very closely coordinated with TG synthesis and appears to be linked to completion of apoB translocation and targeting for secretion. The molecular characteristics of this interaction remain to be determined.