 |
INTRODUCTION |
The assembly and secretion of lipoproteins containing apoB occur
in several tissues (liver, intestine, yolk sac, and heart) and are
essential for the transport and delivery of fat during development and
in the adult (reviewed in Refs. 1-4). There are at least three
distinct human familial disorders associated with developmental and
nutritional abnormalities caused by the inability of the liver and/or
intestine to assemble and secrete apoB-containing lipoproteins (5).
Hypobetalipoproteinemia is caused by mutations of the apoB gene leading
to forms that are incapable of forming lipoprotein particles and/or to
impaired synthesis of apoB by the liver and intestine (1, 6). The
expression of human hypobetalipoproteinemic apoB genes in mice
recapitulates many of the phenotypic abnormalities observed in human
(7, 8). Abetalipoproteinemia is caused by mutations in the
MTP1 gene (9-12). The
absence of MTP lipid transfer activity in the lumen of the endoplasmic
reticulum (ER) causes apoB to be degraded rather than secreted as a
lipoprotein particle by the liver and intestine (12, 13). The recent
demonstration that inactivation of a single allele of the MTP gene in
mice causes significant impairment in the secretion of apoB-containing
lipoproteins by the liver suggests that, in the mouse, MTP plays an
essential role in one or more of the rate-limiting steps in the
lipoprotein assembly/secretion pathway (14). In contrast to mice,
heterozygous abetalipoproteinemic humans appear phenotypically normal
(12, 13). These findings suggest that the role of MTP in regulating lipoprotein assembly/secretion may depend upon complex metabolic relationships that are defined by the genetic background of species and
individuals. The third disorder (chylomicron retention disease) is
unique in that it disables intestinal, but not hepatic, lipoprotein secretion (5). Although the gene defect responsible for chylomicron retention disease remains unknown, its recessive mode of inheritance (5) suggests that its phenotype is caused by a loss-of-function mutation.
Based on what is known about the chylomicron retention disease
phenotype, we have considered the possibility that hepatic and
intestinal lipoprotein assembly/secretion pathways may have distinct
requirements. This interpretation is contrary to studies indicating
that apoB and MTP are the only tissue-specific gene products required
for reconstituting the lipoprotein assembly/secretion pathway in
somatic cells. (In COS-1 cells (15, 16) and HeLa cells (17), which
normally do not express MTP, plasmid-driven expression of apoB and MTP
is sufficient to allow the secretion of apoB-containing lipoproteins,
albeit inefficiently.)
We developed a somatic Chinese hamster ovary (CHO) cell model that
reflects many of the characteristics that are thought to occur in the
livers of abetalipoproteinemic patients. Like the livers of
abetalipoproteinemics, CHO cells do not express MTP and display blocked
translocation of apoB53 into the lumen of the endoplasmic reticulum
(18). The incompletely translocated apoB53 is rapidly degraded by an
ALLN-inhibitable process (18). Recent studies showed that the
expression of liver-specific 7
-hydroxylase (cholesterol,NADPH:oxygen
oxidoreductase (7
-hydroxylating), EC 1.14.13.17) profoundly
influenced the processing of apoB53 by CHO cells (i.e. the
ALLN-inhibitable ubiquitin-dependent proteasome degradation
of apoB was blocked in these cells (designated as JF7 cells) (19)).
Additional studies showed that the translocation-arrested apoB was
N-linked and glycosylated (18) and assumed an orientation in
which 69 kDa of the N terminus of apoB was in the lumen, whereas the
C-terminal portion(s) was exposed to the cytoplasm (20). In the absence
of ALLN, proteolysis caused the N terminus to be released from the ER
membrane, to resume translocation, and to become secreted as an 85-kDa
N-terminal apoB peptide without a lipid core (i.e.
density > 1.21 g/ml) (20). Identical truncated N-terminal apoB
peptides are enriched ~2000-fold in the plasma of
abetalipoproteinemics, leading to the proposal that CHO cells express a
phenotype similar to that of the livers of abetalipoproteinemics (21).
In this study, we examined how complementation of CHO cells with MTP
with or without liver-specific gene 7
-hydroxylase affected the
cellular processing of apoB. The results provide new insights into how
phenotypic gene expression can influence MTP function and the
apoB-containing lipoprotein assembly/secretion pathway.
 |
MATERIALS AND METHODS |
Cell Culture--
B53 cells were cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) containing 1.5%
fetal bovine serum, 3.5% enriched calf serum, and antibiotics as
described (18).
Transfections--
B53 cells (18) were cotransfected with an
expression plasmid encoding rat 7
-hydroxylase driven by the
constitutive cytomegalovirus promoter (22) and a plasmid conferring
resistance to hygromycin (23) in a 12:1 molar ratio. Single cell clones
of cells selected for resistance to 500 µg/ml hygromycin
(i.e. JF7 cells) were found to express 7
-hydroxylase
mRNA, protein, and enzyme activity that were comparable to those of
rat liver (19). B53 cells were cotransfected with a pcDNA3 plasmid
(Invitrogen) that was constructed to contain a cDNA encoding
hamster MTP (a gift from David Gordon) and a plasmid conferring
puromycin resistance in a 12:1 ratio. Cells were selected for
resistance to puromycin (500 µg/ml). Three independent clones were
grown out and shown to express MTP mRNA and protein and to process
apoB as described by the cells designated as B53+MTP cells. JF7 cells
were cotransfected with the pcDNA3 plasmid encoding hamster MTP and
a plasmid conferring puromycin resistance in a 12:1 ratio as described
above. Cells were selected for resistance to puromycin (500 µg/ml).
Three independent clones were grown out and shown to express MTP
mRNA and protein and to process apoB as described by the cells
designated as JF7+MTP cells.
Western Blotting--
Cells were grown to 85% confluence and
treated with ALLN (50 µg/ml) in Me2SO as described in the
figure legends. An equivalent volume of Me2SO was added to
untreated cells. The cells and medium were harvested in
phosphate-buffered saline containing the following protease inhibitors:
aprotinin (100 µg/ml), leupeptin (100 µg/ml), and
phenylmethylsulfonyl fluoride (100 µM). Western blotting
was performed as described (20) using monoclonal antibody 1D1, which recognizes an N-terminal apoB epitope (24).
Northern Blotting--
Cells were grown to 85% confluence on
150-mm dishes. mRNA was obtained and Northern-blotted as described
(25).
Isolation of Microsomal Fractions--
Cells were harvested as
described above and suspended in homogenization buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, and 0.1 mM leupeptin.) The cells were transferred to a nitrogen
decavitation minibomb chamber (Kontes), and the cells were cavitated by
subjecting them to 500 p.s.i. for 20 min at 4 °C and then
rapidly releasing the pressure at atmosphere. The disrupted cell
homogenate was centrifuged for 5 min at 1000 rpm to remove unlysed
cells. The supernatant was then centrifuged for 20 min at 10,000 rpm in
a Beckman J2-20 centrifuge using a JA-20 rotor. The supernatant from
this spin was aliquoted into fresh tubes and centrifuged for 2 h
at 45,000 rpm in a Beckman TL-45 rotor. The supernatant was removed,
and the microsomal pellet was used for immunoprecipitation.
Immunoprecipitation--
Cells were washed with cold
phosphate-buffered saline. Cellular proteins were then solubilized in 1 ml of TETN buffer (25 mM Tris, pH 7.5, 5 mM
EDTA, 1% Triton X-100, and 250 mM NaCl) containing the
following protease inhibitors: aprotinin (100 µg/ml), leupeptin (100 µg/ml), and phenylmethylsulfonyl fluoride (100 µM).
Protein A-Sepharose beads were preincubated with unlabeled cell extract
overnight at 4 °C. A rabbit antiserum specific for human apoB (5 µl) was then incubated with 20 µl of protein A-Sepharose beads (dry
volume) in 1 ml of TETN buffer overnight at 4 °C. The antibody-bound
protein A-Sepharose conjugates were incubated at 4 °C with the
labeled cellular protein samples. Beads were recovered by
centrifugation and washed three times in ice-cold TETN buffer. The
immunoprecipitates were then rinsed with phosphate-buffered saline,
dissolved in 100 µl of sample buffer containing SDS and
-mercaptoethanol, and separated on a 1-20% gradient
SDS-polyacrylamide gel.
Isolation of [35S]Methionine-labeled
Lipoproteins--
B53, JF7, B53+MTP, and JF7+MTP cells were cultured
to 80% confluence and then incubated with oleic acid conjugated to
albumin (0.5 mM) and [35S]methionine (100 µCi/ml) for 24 h. The medium was harvested in the presence of
the following proteolytic inhibitors: aprotinin (100 µg/ml),
leupeptin (100 µg/ml), phenylmethylsulfonyl fluoride (100 µM), and EDTA (0.1%). The medium was then centrifuged
for 5 min at 1000 rpm to remove any cells. The medium samples were adjusted to a density of 1.065 g/ml with KBr and subjected to ultracentrifugation in a Beckman L8 centrifuge using an SW 40 rotor at
35,000 rpm for 48 h at 5 °C. The lipoprotein fractions were
then obtained by tube slicing and desalted using Sepharose CL-4B beads.
Fractions containing radioactivity were separated via SDS-PAGE and
analyzed by autoradiography.
MTP-facilitated Lipid Transfer Activity--
The MTP lipid
transfer assay was similar to the procedure established by Wetterau and
Zilversmit (26) with minor modifications (14). Cells were homogenized
in a buffer containing 50 mM KCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, and protease inhibitor mixture (Boehringer Mannheim). Samples were diluted to 1.75 mg/ml total cell
protein, and deoxycholate was added to a final concentration of 0.05%.
After a 30-min incubation on ice, samples were centrifuged at
100,000 × g for 1 h at 4 °C. The supernatant,
containing the luminal contents (i.e. MTP), was pipetted off
and dialyzed against sample buffer (15 mM Tris, pH 7.4, 40 mM NaCl, and 1 mM EDTA). Donor vesicles (40 nM egg phosphatidylcholine, 0.08 nM
[14C]triolein (NEN Life Science Products), and 3.0 nM cardiolipin) and acceptor vesicles (240 nM
egg phosphatidylcholine and 0.48 nM triolein) were prepared
in sample buffer. Aliquots of the cell extracts were added to the
vesicle preparation and assayed for triglyceride transfer activity at
37 °C in sample buffer as described (14, 26). The reaction was
terminated by adding DEAE-cellulose beads. After pelleting the
DEAE-bound donor vesicles, triglyceride transfer was quantitated by
measuring the radioactivity in the supernatant. Transfer activity was
calculated as percentage triglyceride transferred per h and 100 µg of
total cell protein assuming first-order kinetics (14, 26).
[14C]Acetate Labeling--
Cells were incubated in
serum-containing culture medium with [2-14C]acetate (5 µCi, specific activity of 47 mCi/mol) for 1 h. Cells were
harvested by scrapping them off the dish with a rubber policeman and
centrifuged at 400 × g for 10 min, and the pellet was
used for extraction and protein assay. The culture medium was
centrifuged at 400 × g for 10 min to remove cell
debris. Cells and medium were extracted with chloroform/methanol and
separated on silica gel TLC plates, and the silica gel containing the
individual lipid classes was scraped into scintillation vials for
radioactivity quantitation using
-scintillation counting (27).
Statistical Analysis--
Results are given as means ± S.D. Statistical analysis was determined by Student's t
test. Values of p
0.05 were considered to be significant.
 |
RESULTS |
Stable Expression of Cholesterol 7
-Hydroxylase and MTP in CHO
Cells Affects the Intracellular Fate of ApoB53 in CHO
Cells--
Similar to previous results (18, 20), in the absence of the
proteolytic inhibitor ALLN, CHO cells expressing apoB53 mRNA (B53
cells) contained little apoB53 (Fig. 1,
lane 1). Adding ALLN caused intact apoB53 to accumulate in
B53 cells (Fig. 1, lane 2). JF7 cells, which as a result of
expressing 7
-hydroxylase have a block in the
ubiquitin-dependent proteasome degradation of
translocation-arrested apoB53 (19), showed levels of intact apoB53
(Fig. 1, lane 3) that were indistinguishable from those of
B53 cells treated with ALLN (Fig. 1, lane 2). Similar to
previous results (19), treating JF7 cells with ALLN did not increase the intracellular content of apoB53 (Fig. 1, lane 4).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of cholesterol
7 -hydroxylase and MTP on the fate of apoB in
CHO cells. B53, JF7, B53+MTP, and JF7+MTP cells were plated and
grown to 85% confluence with (+) or without ( ) ALLN (50 µg/ml) for
14 h prior to harvest. Cell extracts (50 µg protein) were
separated by SDS-PAGE, Western-blotted, and detected (using ECL) with
mouse monoclonal antibody 1D1, recognizing an N-terminal epitope.
|
|
To investigate the effect of MTP on apoB processing in CHO cells,
hamster MTP was expressed in B53 and JF7 cells. Unexpectedly, B53 cells
stably expressing MTP (B53+MTP cells) showed no detectable level of
intact apoB53 (Fig. 1, lane 5), suggesting that it was degraded. Furthermore, treating these cells with the proteolytic inhibitor ALLN did not cause intact apoB to accumulate (Fig. 1, lane 6). Stable expression of MTP in JF7 cells (JF7+MTP
cells) did not affect the intracellular content of intact apoB53 either in the absence (Fig. 1, lane 7) or presence (lane
8) of ALLN. These results were found in three individual single
clones of each type of stably expressing CHO cells (data not shown) and thus are not a unique characteristic of a single clone.
In CHO Cells, MTP Requires the Coexpression of 7
-Hydroxylase to
Secrete Intact ApoB53 as a Lipoprotein Particle--
Similar to
previous results (18, 20), either in the absence (Fig.
2, lane 1) or presence
(lane 2) of ALLN, no intact apoB53 was secreted into the
culture medium by B53 cells. Although there was a marked accumulation
of intact apoB53 in JF7 cells (Fig. 1, lanes 3 and
4), no detectable apoB53 was secreted into the culture
medium by JF7 cells treated with and without ALLN (Fig. 2, lanes
3 and 4).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of cholesterol
7 -hydroxylase and MTP on the secretion of apoB
in CHO cells. B53, JF7, B53+MTP, and JF7+MTP cells were plated and
grown to 85% confluence with (+) or without ( ) ALLN (50 µg/ml) for
14 h prior to harvest. Media samples were separated by SDS-PAGE,
Western-blotted, and detected (using ECL) with mouse monoclonal
antibody 1D1, recognizing an N-terminal epitope.
|
|
B53 cells expressing MTP (B53+MTP cells) also did not secrete intact
apoB53 either in the absence (Fig. 2, lane 5) or presence (lane 6) of ALLN. However, JF7 cells expressing MTP (JF7+MTP
cells) did secrete intact apoB53 (Fig. 2, lane 7). ALLN did
not affect the amount of intact apoB53 secreted by these cells (Fig. 2,
lane 8).
Further analysis of the apoB53 secreted by JF7+MTP cells and incubated
with oleic acid (1 mM) showed that it had a density <1.065
g/ml (Fig. 3). These data suggest that
coexpression of 7
-hydroxylase and MTP allows CHO cells to assemble
and secrete apoB-containing lipoproteins.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Secretion of apoB53 as a lipoprotein particle
by JF7+MTP cells. Cells were incubated with oleic acid (1 mM) and [35S]methionine (100 µCi/ml) for
24 h. The medium was adjusted to a density of 1.065 g/ml with KBr
and subjected to ultracentrifugation. The lipoprotein fraction was
obtained by tube slicing. Samples were fractionated using Sephadex G-25
beads. Fractions with radioactivity were subjected to SDS-PAGE, and
radioactivity was assayed using a PhosphorImager (Molecular Dynamics,
Inc., Sunnyvale, CA).
|
|
In CHO Cells, MTP-facilitated Lipid Transfer Activity Requires
7
-Hydroxylase--
In both groups of cells transfected with the MTP
expression plasmid, the levels of both MTP mRNA (Fig.
4A) and protein (Fig. 4B) were similar. Thus, differences in the expression of MTP
protein cannot account for the differences between B53+MTP and JF7+MTP cells in secreting apoB-containing lipoproteins. In CHO cells not
transfected with the MTP expression plasmid, there were no detectable
levels of MTP mRNA (Fig. 4A) or protein (Fig.
4B).

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 4.
Expression of microsomal triglyceride
transfer protein mRNA (A) and protein
(B) in CHO cells. B53, JF7, B53+MTP, and JF7+MTP
cells were plated and grown to 85% confluence in medium without
antibiotics, harvested, and extracted for poly(A)+ RNA and
protein. A, poly(A)+ RNA was Northern-blotted
and probed for MTP and -actin. B, protein extracts were
Western-blotted and probed with an affinity-purified rabbit antiserum
made against a synthetic peptide to MTP. The blots were then stripped
and reprobed with a specific antibody against protein-disulfide
isomerase (PDI).
|
|
MTP-facilitated lipid transfer activity was essentially absent in
extracts from B53, JF7, or B53+MTP cells (Fig.
5). In marked contrast, fairly high
levels of MTP-facilitated lipid transfer activity were present in
extracts of JF7+MTP cells (Fig. 5). The level of MTP lipid transfer
activity expressed by JF7+MTP cells was similar to the activity
observed in primary cultured mouse hepatocytes (Fig. 5). These findings
were obtained with four separate preparations of cell extracts from
four individual plates of cells. Thus, there was a concordance in the
ability of MTP to allow the secretion of apoB-containing lipoproteins
and the enzymatic lipid transfer activity of cell extracts (compare
results in Fig. 3 with the results in Fig. 5).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Analysis of microsomal triglyceride transfer
protein-facilitated lipid transfer activity. A, B53,
JF7, B53+MTP, and JF7+MTP cells and primary cultured mouse hepatocytes
were harvested and analyzed for MTP lipid transfer activity using the
procedure established by Wetterau and Zilversmit (26) with minor
modifications (14). Values represent the means ± S.D. of three
separate cell extracts in each group of cells. BSA
represents the lipid transfer activity without the cell extracts
(background). This experiment was repeated three additional times
(using different plates of cells); the results were similar to those
shown. Wild-type CHO-K1 cells displayed the same level of MTP lipid
transfer activity as B53 cells. B, extracts from JF7+MTP
cells were assayed for MTP lipid transfer activity in undiluted
samples, in samples that were diluted 50% with buffer, and in samples
that were diluted 50% with extracts from B53+MTP cells. Values
represent the means ± S.D. of three separate cell extracts in
each group of cells. This experiment was repeated three additional
times (using different plates of cells); the results were similar to
those shown. TG, triglyceride.
|
|
To examine the possibility that, in B53+MTP cells, the lack of
MTP-facilitated lipid transfer activity was due to the presence of an
inhibitor, we performed mixing experiments (Fig. 5). Adding extracts of
B53+MTP cells to extracts from JF7+MTP cells caused the same
"dilution" of activity as adding buffer alone (Fig. 5). These data
indicate that the inability of B53+MTP cells to display MTP-facilitated
lipid transfer activity is not likely to be caused by the presence of
an inhibitor that can readily associate with or dissociate from MTP in
the assay buffer.
In B53 Cells, Expression of MTP Allows ApoB to be Translocated into
the ER and Subsequently Targeted to Degradation by a DTT- and
Chloroquine-inhibitable Process--
It is now established that ALLN
inhibits the degradation of translocation-arrested apoB by blocking
ubiquitin-dependent proteasome degradation (19, 28-31).
ALLN also inhibits the degradation of apoB in the lumen of the ER by
blocking ER-60 protease (32). The inability of ALLN to block the
degradation of apoB53 in B53 cells expressing MTP (Fig. 1, lane
6) suggests that it is degraded in an intracellular compartment in
which apoB degradation is not dependent upon ALLN-inhibitable
proteases. There are at least two additional proteolytic processes that
have been reported to degrade apoB and that are resistant to inhibition
by ALLN: 1) a DTT-inhibitable process that degrades apoB, which is
completely translocated into the ER lumen (33); and 2) a
chloroquine-inhibitable protease that resides in the
lysosomal/endosomal compartment (34). To examine if the apoB53
expressed by B53+MTP cells was degraded by either of these proteolytic
process, these cells were labeled with [35S]methionine to
detect intact apoB53 in the absence and presence of chloroquine (100 µm) and DTT (1 mM) for 4 h. Immunoprecipitation and
autoradiography showed that there was an ~8-fold increase in the
amount of intact apoB53 in the whole cell extract of cells treated with
DTT and an ~20-fold increase in the amount of intact apoB53 in the
whole cell extract of cells treated with chloroquine compared with
untreated cells (Fig. 6A).
Ultracentrifugation of the whole cell extract followed by
immunoprecipitation showed that most (>85%) of the intact apoB53 that
accumulated in cells treated with chloroquine was in the 10,000 × g pellet (Fig. 6B). The amount of intact apoB53
that accumulated in the microsomal fraction (100,000 × g pellet) was ~5% of the amount in the 10,000 × g pellet. No detectable intact apoB53 was found in the
cytoplasm (data not shown). These data suggest that, in B53+MTP cells,
a significant portion of apoB53 enters the ER lumen of the secretory pathway and is transported to an acidic compartment (i.e.
lysosomes and endosomes), where it is degraded.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of DTT and chloroquine on the
accumulation of apoB53 in B53 cells expressing MTP (A)
and the accumulation of apoB53 in subcellular membrane fractions
prepared from cells treated with chloroquine (B).
A, B53+MTP cells were grown to 85% confluence. Cells were
incubated with culture medium containing [35S]methionine
(400 µCi/ml) with or without DTT (1 mM) or chloroquine
(100 µM) for 4 h. Aliquots were immunoprecipitated,
separated by SDS-PAGE, and developed using a PhosphorImager. The
analysis of duplicate plates of cells is shown for each culturing
condition. B, B53+MTP cells were grown to 85% confluence.
Cells were incubated with culture medium containing
[35S]methionine (400 µCi/ml) with or without
chloroquine (100 µM) for 4 h. Cells were scraped off
the culture dishes, disrupted by nitrogen cavitation, and separated
into membrane fractions by ultracentrifugation (49). Equal aliquots of
each fraction were immunoprecipitated, separated by SDS-PAGE, and
developed using a PhosphorImager.
|
|
Coexpression of 7
-Hydroxylase with MTP Increases the Rate of
Synthesis of Lipoprotein Lipids--
In McArdle rat hepatoma cells,
plasmid-driven expression of 7
-hydroxylase increased the synthesis
and secretion of all very low density lipoprotein lipids (22). We
examined if 7
-hydroxylase may have affected lipid synthesis in CHO
cells by measuring the incorporation of [14C]acetate into
cellular and secreted phospholipids, triglycerides, cholesterol, and
cholesterol esters (Fig. 7). In B53+MTP
cells, the synthesis of phospholipids, triglycerides, cholesterol, and cholesterol esters was significantly less than that in JF7+MTP cells
(Fig. 7A). The decrease in lipid biosynthesis displayed by
B53+MTP cells was further demonstrated by a decrease in the secretion
of these lipids (Fig. 7B).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Lipid synthesis (A) and
secretion (B) by CHO cells expressing microsomal
triglyceride transfer protein with and without
7 -hydroxylase. B53+MTP and JF7+MTP cells
were plated and grown to 85% confluence. [2-14C]Acetate
(3.3 µCi/ml) was added to the medium, and cells were incubated for
2 h. Cells were then harvested; radiolabeled lipids were separated
by TLC; and the radioactivity was quantitated by scintillation
analysis. Open bars represent CHO cells expressing apoB53
and MTP, but no 7 -hydroxylase. Closed bars represent CHO
cells expressing apoB53, MTP, and 7 -hydroxylase. Values represent
the means ± S.D. of three separate cell extracts in each group of
cells.
|
|
 |
DISCUSSION |
Our results show for the first time that, in CHO cells, MTP plays
two distinct roles in the cellular processing of apoB: 1) facilitating
translocation across the ER and 2) providing lipid transfer for the
assembly and secretion of apoB-containing lipoprotein particles.
Although both functions are required to assemble and secrete
apoB-containing lipoproteins, they do not occur in a concerted manner
in CHO cells expressing MTP protein (i.e. without
7
-hydroxylase, translocation occurs, whereas lipid transfer does
not). Based on these unique findings, we propose that MTP functions as
an intraluminal "chaperone" to facilitate apoB translocation and as
a lipid transfer protein in the assembly of apoB-containing lipoproteins.
Several lines of evidence indicate that, by itself, in the absence of
7
-hydroxylase expression, MTP releases apoB from translocation arrest in CHO cells. First, cells expressing MTP without
7
-hydroxylase display no ALLN-induced increase in the cellular
content of intact apoB53 (Fig. 1). Since, in the absence of MTP
expression in B53 cells, ALLN blocks the degradation of
translocation-arrested apoB53 (20), these findings suggest that MTP
removed apoB53 from this first step in the secretory pathway. Second,
in B53 cells expressing MTP without 7
-hydroxylase, the lysosomal
inhibitor chloroquine blocks the degradation of apoB53 (Fig. 6). As a
result, apoB53 accumulates in the 10,000 × g fraction,
suggesting that at least a portion of the apoB53 that has entered the
ER lumen can be transported to a compartment containing acidic
(lysosomal) proteases. In marked contrast, essentially all of the
intact apoB53 that accumulates in ALLN-treated B53 cells not expressing
MTP is in the microsomal membrane fraction (i.e.
100,000 × g fraction) in a form that is both
glycosylated and susceptible to trypsin digestion, suggesting that it
has a transmembrane, translocation-arrested orientation in the ER and
cannot move further through the secretory pathway (18, 20). The
simplest interpretation of these combined data is that expressing MTP
in B53 cells allows intact apoB to be completely translocated across
the ER membrane and to enter the secretory pathway. However, in the
absence of 7
-hydroxylase expression, B53 cells expressing MTP do not
display detectable MTP-facilitated transfer activity and lipoprotein
assembly, and secretion remains blocked. In marked contrast, JF7 cells
expressing MTP both display MTP-facilitated lipid transfer activity and
secrete apoB-containing lipoproteins. These combined data indicate
that, in CHO cells, MTP-facilitated translocation and MTP-facilitated
lipid transfer are separable functions and that both are required for
the efficient assembly and secretion of apoB-containing lipoproteins.
Our conclusions that MTP functions to facilitate both apoB
translocation and the addition of lipid to the nascent lipoprotein particle are consistent with results obtained from studies using chemical inhibitors of MTP lipid transfer. Chemical inhibition of MTP
lipid transfer activity has been reported to impair the assembly and
secretion of apoB-containing lipoproteins by blocking an early event in
the secretory pathway (30, 35-38). Additional pulse-chase studies in
HepG2 cells suggested that chemical inhibition of MTP lipid transfer
blocks the translocation of apoB100, causing it be cotranslationally
degraded by a process attributed to the proteasome (30). These
conclusions were further supported by studies showing that inhibition
of MTP results in translocation arrest and rapid cotranslational
degradation of apoB100 by a ubiquitin-dependent proteasome
process (39). In addition to blocking apoB translocation, inhibition of
MTP lipid transfer also impairs a later process in the secretory
pathway (i.e. the addition of lipid to form a more fully
lipidated lipoprotein particle). In separate studies using McArdle rat
hepatoma cells, chemical inhibition of MTP lipid transfer activity was
shown to block the oleic acid stimulation of apoB48 secretion (40) and
the formation and secretion of very low density lipoprotein containing
apoB100 (41).
The combined data suggest that MTP may act as a multifunctional
chaperone via both lipid transfer-independent and
-dependent processes. MTP lipid transfer-independent
processes, which occur in B53+MTP cells, may facilitate the folding and
cotranslational disulfide formation of apoB (16, 42, 43). MTP lipid
transfer-dependent processes may also facilitate apoB
folding in a manner that may be coupled to lipoprotein particle
formation (40, 41, 44).
Previous studies showed that transfecting HeLa and COS cells with MTP
expression plasmids, similar to one we used in our studies, results in
MTP lipid transfer activity and the assembly and secretion of
apoB-containing lipoproteins (15, 17). In other studies using
mammary-derived mouse cells (C127), which do not express MTP,
apoB-containing lipoproteins were assembled and secreted (45). In
contrast, our findings indicate that CHO cells require both MTP and
7
-hydroxylase to display MTP lipid transfer activity and the
assembly and secretion of apoB53-containing lipoprotein particles.
These combined results suggest that factors present in each cell type
and experimental system profoundly influence the requirements for the
translocation and processing of apoB and lipid components into
lipoprotein particles.
There are sufficient data to indicate that 7
-hydroxylase
significantly influences several processes that, depending upon the
cell phenotype, may be rate-limiting in the lipoprotein
assembly/secretion pathway. In McArdle rat hepatoma cells, expression
of 7
-hydroxylase increases the cellular content of mature SREBP1 and
the expression of MTP mRNA and protein (22). As a result, McArdle
rat hepatoma cells expressing 7
-hydroxylase become resistant to
oleic acid stimulation of apoB100 secretion (22). In CHO cells, the
coexpression of 7
-hydroxylase with MTP is required for
MTP-facilitated lipid transfer activity (Fig. 5). Expression of
7
-hydroxylase in CHO cells also changes many other phenotypic
characteristics: it increases the synthesis and secretion of all very
low density lipoprotein lipids by CHO cells (Fig. 7), and it blocks
ubiquitin conjugation and subsequent proteasome degradation of
translocation-arrested apoB53 (19). Phenotypic metabolic relationships
that determine the process(es) that may be rate-limiting for
lipoprotein assembly/secretion are likely to include the rate of apoB
synthesis, the rate of lipogenesis, the expression of MTP protein and
lipid transfer activity, and the activity of apoB-degradative
processes. With the proviso that our results obtained studying the
cellular processing of apoB53 in CHO cells may reflect similar
processes that occur in the liver expressing apoB100 and the intestine
expressing apoB48, our findings may provide a new insight into
explaining why intestinal and hepatic processing of apoB may have
different constraints. One of the most provocative differences is
chylomicron retention disease, which blocks the secretion of
apoB-containing lipoproteins by the intestine, but not the liver (5).
Affected patients produce immunoreactive apoB48 in enterocytes, process
apoB mRNA to the intestinal (edited) form, and express MTP
(46-48). These data suggest that the inability of the intestines of
chylomicron retention disease patients to secrete apoB-containing
lipoproteins is due to the functional loss of a gene product other than
apoB or MTP. Our findings showing that the liver-specific enzyme
7
-hydroxylase is required for CHO cells expressing apoB and MTP to
secrete apoB-containing lipoproteins support the possibility that
expression of 7
-hydroxylase in the livers of chylomicron retention
disease patients may complement a defective process that is common to
both intestinal and hepatic lipoprotein assembly/secretion pathways.